Antibody formulation

ABSTRACT

The present invention relates to pharmaceutical formulations of a pharmaceutically active antigen binding protein, for example a monoclonal antibody. Such formulations comprise, in addition to the antigen binding protein, a buffering agent and a tonicity agent.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations of apharmaceutically active antigen binding protein, for example amonoclonal antibody. Such formulations comprise, in addition to theantigen binding protein, a buffering agent and a tonicity agent.

BACKGROUND OF THE INVENTION

The pharmaceutical use of antibodies has increased over the past years.In many instances such antibodies are injected via the intravenous (IV)route. Unfortunately the amount of antibody that can be injected via theintravenous route is limited by the physico-chemical properties of theantibody, in particularly by its solubility and stability in a suitableliquid formulation and by the volume of the infusion fluid. Alternativeadministration pathways are subcutaneous or intramuscular injection,which offer potential advantages in terms of patient compliance and easeof administration. These injection pathways require high proteinconcentration in the final solution to be injected.

Accordingly, there is a desire to provide highly concentrated, stablepharmaceutical formulations of therapeutically active antigen bindingproteins such as antibodies for subcutaneous injection. The advantage ofsubcutaneous injections is that it allows the medical practitioner toperform it in a rather short intervention with the patient. Moreover thepatient can be trained to perform the subcutaneous injection by himself.Such self-administration is particularly useful during maintenancedosing because no hospital care is needed (reduced medical resourceutilization). Usually injections via the subcutaneous route are limitedto approximately 2 mL. For patients requiring multiple doses, severalunit dose formulations can be injected at multiple sites of the bodysurface.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a pharmaceuticalformulation for an antigen binding protein comprising a buffering agentand a tonicity agent. More particularly, the present invention providesabout 150 to 250 mg/mL antigen binding protein; about 1 to 100 mM of abuffering agent providing a pH of about 5.0 to about 7.0; and about 70to 170 mM of a tonicity agent. In one embodiment the antigen bindingprotein is an anti-BLyS antibody.

In another aspect the present invention provides a pharmaceuticalformulation for an antigen binding protein comprising a buffering agent,a stabilizer, a tonicity agent, and a nonionic surfactant. Moreparticularly, the present invention provides a pharmaceuticalformulation comprising the antigen binding protein, histidine, arginine,NaCl, and polysorbate 80. In one embodiment the antigen binding proteinis an anti-BLyS antibody.

In another aspect the present invention provides for a method oftreating a disease or condition which is amenable to treatment with ananti-BLyS antibody in a subject comprising administering a formulationaccording the present invention in a subject in an amount effective totreat the disease or condition. In one aspect the disease or conditionis an autoimmune disease or disorder.

In another aspect the present invention provides for a kit comprisingone or more vials containing the formulation according to the presentinvention and instructions for subcutaneous administration of theformulation to a patient.

In another aspect the present invention provides for an injection devicecomprising a stable anti-BLyS antibody formulation described herein.

In another aspect the present invention provides for a formulationaccording to the present invention for use in the treatment of diseaseselected from the group consisting of systemic lupus erythematosus,anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis, lupusnephritis, primary Sjögren's syndrome, chronic immune thrombocytopenia,myasthenia gravis, symptomatic Waldenström's macroglobulinaemia, immunedesensitizing of patients awaiting kidney transplant, membranousnephropathy, systemic sclerosis, rheumatoid arthritis, multiple myeloma,multiple sclerosis, and kidney failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of protein concentration on aggregation rate forFormulation 1.

FIG. 2 shows the turbidity of Formulations 1 and 5 after 5′ months at2-8° C.

FIG. 3 shows the relationship of belimumab viscosity to concentration.

FIG. 4 shows the change in % aggregate after 3 months storage at 2-8° C.for various formulations.

FIG. 5 shows the change in % aggregate after 3 months storage at varioustemperatures and formulations.

FIG. 6 shows the effect of temperature on aggregation rates after 5months at up to 25° C., and shows that the arginine formulation (opensquares on the graph) significantly dampens aggregation when compared toFormulation 1 (filled squares).

FIG. 7 shows aggregation rates of Formulation 1 (full squares; 06-C) andFormulation 5 (hollow squares; 06-D) between 125 and 200 mg/mL and -80°C. to 40° C. after 5 Months Storage and how consistently Formulation 5(dashed lines) shows a lower aggregation rate than Formulation 1 (solidlines).

FIG. 8 shows reducing CGE degradation rates of formulations 1 (06-C) and5 (06-D) between 125 and 200 mg/mL and -80° C. to 40° C. after 5/monthsstorage.

FIG. 9 shows acidic rates of Formulations 1 (full squares: 06-C) and 5(hollow squares: 06-D) between 125 and 200 mg/mL and -80° C. to 40° C.after 5 months storage.

FIG. 10 shows belimumab heavy chain oxidation levels in Formulations 1and 5 between 125 and 200 mg/mL and -80° C. to 40° C. after 5¼ monthsstorage.

FIG. 11 shows peptide map of belimumab in Formulation 1 at 200 mg/mLafter 5¼ months storage.

FIG. 12 shows peptide map of belimumab in Formulation 5 at 200 mg/mLafter 5¼ months storage.

FIG. 13 shows peptide map of belimumab samples with different argininelevels.

FIG. 14 shows HTF pH-buffer screening.

FIG. 15 shows two factor interaction—pH×buffer—SEC monomer.

FIG. 16 shows two factor interaction—pH×buffer—cIEF main.

FIG. 17 shows viscosity of anti-IL13 antibody at various concentrations.

FIG. 18 shows viscosity (cP) vs. concentration (mg/mL) results foranti-IL13 T=1 samples from shake study.

FIG. 19 shows that 7 day acetate samples were gelled (left). No gel wasobserved in the succinate or histidine samples (center and right). The10 day succinate vial was observed to be in a semi-gel state.

FIG. 20 shows a comparison of near-UV circular dichroism spectra for 3month chemical stability samples.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions, or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an”, and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a polypeptide” includes a combination of twoor more polypeptides, and the like.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

In one aspect the present invention provides a pharmaceuticalformulation for an antigen binding protein comprising a buffering agent,and a tonicity agent. In another aspect the present invention provides apharmaceutical formulation for an antigen binding protein comprising abuffering agent, a stabilizer, a tonicity agent, and a nonionicsurfactant. In one embodiment the formulation is lyophilized or spraydried. In certain embodiments the formulation is lyophilized or spraydried and then later reconstituted with a dispersing agent. In oneembodiment the dispersing agent is sterile water or “water forinjection” (WFI). The antigen binding protein can be further dilutedwith isotonic saline or other excipients to produce a desirableconcentration prior to administration. In one embodiment the formulationis a reconstituted formulation. In another embodiment the formulation isa liquid pharmaceutical formulation.

The term “pharmaceutical formulation” or “formulation” refers to apreparation which is in such form as to permit the biological activityof the active ingredient to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. Such formulations are sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

In exemplary embodiments of the present invention, the liquidformulations exhibit desirable characteristics, such as desirableviscosity and surface tension characteristics.

The term “surface tension” refers to the attractive force exerted by themolecules below the surface upon those at the surface/air interface,resulting from the high molecular concentration of a liquid compared tothe low molecular concentration of the gas. Liquids with low values ofsurface tension, such as nonpolar liquids, flow more readily than water.Typically, values of surface tensions are expressed in newtons/meters ordynes/centimeters.

“Dynamic surface tension” as referred to herein is the surface/airinterface and the dynamic interfacial tension to the surface/surfaceinterface. There are a number of alternative methods for measuringdynamic surface tension, for example, captive bubble surfacetensionometry or pulsating bubble surface tensionometry.

The term “viscosity” refers to the internal resistance to flow exhibitedby a fluid at a specified temperature, the ratio of shearing stress torate of shear. A liquid has a viscosity of one poise if a force of 1dyne/square centimeter causes two parallel liquid surfaces one squarecentimeter in area and one square centimeter apart to move past oneanother at a velocity of 1 cm/second. One poise equals one hundredcentipoise.

In one embodiment, the viscosity of the formulation comprising bufferingagent and stabilizer is reduced by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or atleast about 30% compared to the viscosity of the formulation in theabsence of buffering agent and stabilizer. In one embodiment, theviscosity of the formulation comprising buffering agent and stabilizeris less than about 50 cP, less than about 45 cP, less than about 40 cP,less than about 35 cP, less than about 30 cP, less than about 25 cP,less than about 20 cP, less than about 15 cP, or less than about 10 cP.

When referring to apparent viscosity, it is understood that the value ofviscosity is dependent on the conditions under which the measurement wastaken, such as temperature, the rate of shear and the shear stressemployed. The apparent viscosity is defined as the ratio of the shearstress to the rate of shear applied. There are a number of alternativemethods for measuring apparent viscosity. For example, viscosity can betested by a suitable cone and plate, parallel plate or other type ofviscometer or rheometer.

“Gelation is defined as the process of formation of a stiff networkpresumably caused by the onset of topological overlaps amongpolymerizing mAb or filaments as well as the cross-linking and bundlingof these filaments. This stiff network manifests as a solution elasticmodulus (G) as well as an increase in its inherent viscous modulus(G″).”

In one aspect, the present invention is directed to a method of reducingor inhibiting gelation of a solution comprising utilizing a formulationof the present invention. In another aspect, the present invention isdirected to a method of reducing or inhibiting gelation of a solutioncomprising a therapeutic protein, the method comprising administeringhistidine and sodium chloride to the solution.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. A polypeptide can be ofnatural (tissue-derived) origins, recombinant or natural expression fromprokaryotic or eukaryotic cellular preparations, or produced chemicallyvia synthetic methods. The terms apply to amino acid polymers in whichone or more amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymer. Amino acid mimetics refers to chemical compounds that have astructure that is different from the general chemical structure of anamino acid, but that functions in a manner similar to a naturallyoccurring amino acid. Non-natural residues are well described in thescientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine: D- orL-phenylglycine; D- or L-2 thieneylalanine: D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine:D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine:D-p-fluoro-phenylalanine: D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenylphenylalanine: D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

“Peptide” as used herein includes peptides which are conservativevariations of those peptides specifically exemplified herein.“Conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include, but are not limited to, thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan,tyrosine, norleucine or methionine for another, or the substitution ofone polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. Neutral hydrophilic amino acids which can be substituted forone another include asparagine, glutamine, serine and threonine.“Conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Such conservative substitutions arewithin the definition of the classes of the peptides of the invention.The biological activity of the peptides can be determined by standardmethods known to those of skill in the art and described herein.

“Recombinant” when used with reference to a protein indicates that theprotein has been modified by the introduction of a heterologous nucleicacid or protein or the alteration of a native nucleic acid or protein.

As used herein a “therapeutic protein” refers to any protein and/orpolypeptide that can be administered to a mammal to elicit a biologicalor medical response of a tissue, system, animal or human that is beingsought, for instance, by a researcher or clinician. A therapeuticprotein may elicit more than one biological or medical response.Furthermore, the term “therapeutically effective amount” means anyamount which, as compared to a corresponding subject who has notreceived such amount, results in, but is not limited to, healing,prevention, or amelioration of a disease, disorder, or side effect, or adecrease in the rate of advancement of a disease or disorder. The termalso includes within its scope amounts effective to enhance normalphysiological function as well as amounts effective to cause aphysiological function in a patient which enhances or aids in thetherapeutic effect of a second pharmaceutical agent.

All “amino acid” residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as shown in the followingtable.

TABLE 1 Amino acid abbreviations. 1 Letter 3 Letter Amino Acid Y TyrL-tyrosine G Gly L-glycine F Phe L-phenylalanine M Met L-methionine AAla L-alanine S Ser L-serine I Ile L-isoleucine L Leu leucine T ThrL-threonine V Val L-valine P Pro L-proline K Lys L-lysine H HisL-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp L-tryptophan RArg L-arginine D Asp L-aspartic acid N Asn L-asparagine C CysL-cysteine.

It should be noted that all amino acid residue sequences are representedherein by formulae whose left to right orientation is in theconventional direction of amino-terminus to carboxy-terminus.

In another embodiment the polypeptide is an antigen binding protein. Inone embodiment the antigen binding protein is selected from the groupconsisting of a soluble receptor, antibody, antibody fragment,immunoglobulin single variable domain, Fab, F(ab′)2, Fv, disulphidelinked Fv, scFv, closed conformation multispecific antibody,disulphide-linked scFv, or diabody.

The term “antigen binding protein” as used herein refers to antibodies,antibody fragments and other protein constructs which are capable ofbinding to an antigen.

The terms Fv, Fc, Fd, Fab, or F(ab)2 are used with their standardmeanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, ColdSpring Harbor Laboratory, (1988)).

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., Queen et al.,Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al.,Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may beone selected from a conventional database, e.g., the KABA™ database, LosAlamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanized antibodies—see for example EP-A-0239400 andEP-A-054951.

The term “donor antibody” refers to an antibody (monoclonal, and/orrecombinant) which contributes the amino acid sequences of its variableregions. CDRs, or other functional fragments or analogs thereof to afirst immunoglobulin partner, so as to provide the alteredimmunoglobulin coding region and resulting expressed altered antibodywith the antigenic specificity and neutralizing activity characteristicof the donor antibody.

The term “acceptor antibody” refers to an antibody (monoclonal and/orrecombinant) heterologous to the donor antibody, which contributes all(or any portion, but in some embodiments all) of the amino acidsequences encoding its heavy and/or light chain framework regions and/orits heavy and/or light chain constant regions to the firstimmunoglobulin partner. In certain embodiments a human antibody is theacceptor antibody.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883.

As used herein the term “domain” refers to a folded protein structurewhich has tertiary structure independent of the rest of the protein.Generally, domains are responsible for discrete functional properties ofproteins and in many cases may be added, removed or transferred to otherproteins without loss of function of the remainder of the protein and/orof the domain. An “antibody single variable domain” is a foldedpolypeptide domain comprising sequences characteristic of antibodyvariable domains. It therefore includes complete antibody variabledomains and modified variable domains, for example, in which one or moreloops have been replaced by sequences which are not characteristic ofantibody variable domains, or antibody variable domains which have beentruncated or comprise N- or C-terminal extensions, as well as foldedfragments of variable domains which retain at least the binding activityand specificity of the full-length domain.

The phrase “immunoglobulin single variable domain” refers to an antibodyvariable domain (V_(H), V_(HH), V_(L)) that specifically binds anantigen or epitope independently of a different V region or domain. Animmunoglobulin single variable domain can be present in a format (e.g.,homo- or hetero-multimer) with other, different variable regions orvariable domains where the other regions or domains are not required forantigen binding by the single immunoglobulin variable domain (i.e.,where the immunoglobulin single variable domain binds antigenindependently of the additional variable domains). A “domain antibody”or “dAb” is the same as an “immunoglobulin single variable domain” whichis capable of binding to an antigen as the term is used herein. Animmunoglobulin single variable domain may be a human antibody variabledomain, but also includes single antibody variable domains from otherspecies such as rodent (for example, as disclosed in WO 00/29004), nurseshark and Camelid V_(HH) dAbs (nanobodies). Camelid V_(HH) areimmunoglobulin single variable domain polypeptides that are derived fromspecies including camel, llama, alpaca, dromedary, and guanaco, whichproduce heavy chain antibodies naturally devoid of light chains. SuchVan domains may be humanized according to standard techniques availablein the art, and such domains are still considered to be “domainantibodies” according to the invention. As used herein “V_(H) includescamelid V_(HH) domains. NARV are another type of immunoglobulin singlevariable domain which were identified in cartilaginous fish includingthe nurse shark. These domains are also known as Novel Antigen Receptorvariable region (commonly abbreviated to V(NAR) or NARV). For furtherdetails see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.

The term “Epitope-binding domain” refers to a domain that specificallybinds an antigen or epitope independently of a different V region ordomain, this may be a domain antibody (dAb), for example a human,camelid or shark immunoglobulin single variable domain.

As used herein, the term “antigen-binding site” refers to a site on aprotein which is capable of specifically binding to antigen, this may bea single domain, for example an epitope-binding domain, or it may bepaired V_(H)/V_(L) domains as can be found on a standard antibody. Insome aspects of the invention single-chain Fv (ScFv) domains can provideantigen-binding sites.

The terms “mAbdAb” and dAbmAb” are used herein to refer toantigen-binding proteins of the present invention. The two terms can beused interchangeably, and are intended to have the same meaning as usedherein.

The pharmaceutical formulation of the present invention provides about150 to 250 mg/mL antigen binding protein; about 1 to 100 mM of abuffering agent providing a pH of about 5.0 to about 7.0; and about 70to 170 mM of a tonicity agent. Alternatively, the pharmaceuticalformulation of the present invention provides about 150 to 250 mg/mLantigen binding protein; about 1 to 100 mM of a buffering agentproviding a pH of 6.0±0.5; about 1 to 100 mM of a stabilizer; about 90to 150 mM of a tonicity agent; and about 0.005 to 0.015% (w/v) of anonionic surfactant. In one embodiment the antigen binding protein is ananti-B Lymphocyte Stimulator (anti-BLyS) protein antibody.

Also described is a pharmaceutical formulation comprising about 150 to250 mg/mL antigen binding protein; about 1 to 100 mM histidine at pH of6.0±0.5; about 70 to 170 mM NaCl. In one embodiment the formulationfurther comprises about 0.005 to 0.03% (w/v) of a nonionic surfactant.In one embodiment the formulation further comprises about 0.01 to about0.1 mM of a metal chelator. In one embodiment the antigen bindingprotein is an anti-IL-13 antibody.

The pharmaceutical formulation of the present invention may be providedin liquid form or may be provided in lyophilized form.

The pharmaceutical formulation according to the present inventioncomprises a buffering agent. Buffering agents include, but are notlimited to citric acid, HEPES, histidine, potassium acetate, potassiumcitrate, potassium phosphate (KH₂PO₄), sodium acetate, sodiumbicarbonate, sodium citrate, sodium phosphate (NAH₂PO₄). Tris base, andTris-HCl. In one embodiment, the buffering agent is histidine. Incertain embodiments, the histidine concentration is about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM.In one embodiment the histidine concentration is 10±5 mM. In oneembodiment, the histidine concentration is 10-2 mM. In one embodiment,the histidine concentration is about 10 mM. In one embodiment, thehistidine concentration is about 15 mM.

As used herein the term “buffering agent providing a pH of about 5.0 toabout 7.0” refers to an agent which provides that the solutioncomprising it resists changes in pH by the action of its acid/baseconjugate components. The buffer used in the formulations in accordancewith the present invention may have a pH in the range from about 5.5 toabout 6.5, or from about 5.8 to about 6.2. In one embodiment the pH isabout 6.0. In one embodiment the pH is about 6.250. Examples ofbuffering agents that will control the pH in this range include acetate,succinate, gluconate, histidine, citrate, glycylglycine and otherorganic acid buffers. The most suitable buffer in accordance with thepresent invention is a histidine buffer, such as e.g. L-histidine.

A “histidine buffer” is a buffer comprising the amino acid histidine.Examples of histidine buffers include histidine chloride, histidineacetate, histidine phosphate, histidine sulfate. The histidineformulation identified in the examples as being most suitable is ahistidine buffer made from 0.65 mg/mL L-histidine, 1.2 mg/mL L-histidinemonohydrochloride.

The pharmaceutical formulation according to the present inventioncomprises a tonicity agent. Tonicity agents, include, but are notlimited to dextrose, glycerin, mannitol, potassium chloride, and sodiumchloride. In one embodiment the tonicity agent is sodium chloride. Inone embodiment the sodium chloride concentration is about 70 to 170 mM;about 90-150 mM; or about 115±10 mM. In certain embodiments the sodiumchloride concentration is about 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 mM.In one embodiment, the sodium chloride concentration is about 115 mM. Inanother embodiment, the sodium chloride concentration is 150±10 mM. Inone embodiment, the sodium chloride concentration is about 150 mM.

By “isotonic” is meant that the formulation has essentially the sameosmotic pressure as human blood. Isotonic formulations will generallyhave an osmotic pressure from about 250 to 350 mOsm. Isotonicity can bemeasured using a vapor pressure or freezing-point depression typeosmometer.

In certain embodiments the pharmaceutical formulation according to thepresent invention comprises a stabilizer. Stabilizers, include, but arenot limited to human serum albumin (hsa), bovine serum albumin (bsa),α-casein, globulins, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin,and RNase A. Stabilizers also include amino acids and their metabolites,such as, glycine, alanine (α-alanine, β-alanine), arginine, betaine,leucine, lysine, glutamic acid, aspartic acid, proline,4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines(alanopine, octopine, strombine), and trimethylamine N-oxide (TMAO). Inone embodiment the stabilizer is an amino acid. In one embodiment theamino acid is arginine. In one embodiment the arginine concentration isabout 20 to 30 mM. In one embodiment, the arginine concentration isabout 25±2 mM.

In certain embodiments the pharmaceutical formulation according to thepresent invention comprises a nonionic surfactant. Nonionic surfactants,include, but are not limited to, polyoxyethylensorbitan fatty acidesters (such as polysorbate 20 and polysorbate 80),polyethylene-polypropylene copolymers, polyethylene-polypropyleneglycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, e.g.polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), sodium dodecyl sulphate (SDS). In one embodiment the nonionicsurfactant is polysorbate 80. In one embodiment the polysorbate 80concentration is about 0.005 to 0.02% (w/v). In one embodiment, thepolysorbate 80 concentration is about 0.01% (w/v). In one embodiment,the polysorbate 80 concentration is about 0.02% (w/v).

In certain embodiments the pharmaceutical formulation according to thepresent invention comprises a metal chelator. Metal chelators, include,but are not limited to EDTA and EGTA. In one embodiment the metalchelator is EDTA. In one embodiment the EDTA concentration is about 0.01to about 0.02 mM. In one embodiment, the EDTA concentration is about0.05 mM.

In one embodiment, the antigen binding protein is a monoclonal antibodyor fragment thereof. In one embodiment, the monoclonal antibody orfragment thereof is mouse, chimeric, humanized, or fully human. In oneembodiment, the monoclonal antibody or fragment thereof binds to BLyS orIL-13.

In one aspect the formulation comprises the antigen binding protein,histidine, arginine, NaCl, and polysorbate 80. In another aspect theformulation comprises about 200 mg/mL antigen binding protein, about 10mM histidine, about 25 mM arginine, about 115 mM NaCl, and about 0.01%polysorbate 80, at about pH 6.0. In one embodiment, the antigen bindingprotein binds to BLyS.

In one embodiment the pharmaceutical formulation of the presentinvention provides about 200 mg/mL antigen binding protein; about 15 mMhistidine at a pH of about 6.25; about 150 mM NaCl; about 0.02% (w/v)polysorbate 80; and about 0.05 mM EDTA. In one embodiment, the antigenbinding protein binds to IL-13.

In one aspect the pharmaceutical formulation of the present invention isstable upon freezing and thawing. A “stable” formulation is one in whichall the protein therein essentially retain their physical stabilityand/or chemical stability and/or biological activity upon storage at theintended storage temperature, e.g. 2-8° C. It is desired that theformulation essentially retains its physical and chemical stability, aswell as its biological activity upon storage. The storage period isgenerally selected based on the intended shelf-life of the formulation.Furthermore, the formulation should be stable following freezing (to,e.g., −70° C.) and thawing of the formulation, for example following 1,2 or 3 cycles of freezing and thawing. Various analytical techniques formeasuring protein stability are available in the art and are reviewed inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. DrugDelivery Rev. 10: 29-90 (1993), for example. Stability can be measuredat a selected temperature for a selected time period. Stability can beevaluated qualitatively and/or quantitatively in a variety of differentways, including evaluation of aggregate formation (for example usingsize exclusion chromatography, by measuring turbidity, and/or by visualinspection); by assessing charge heterogeneity using cation exchangechromatography or capillary zone electrophoresis; amino-terminal orcarboxy-terminal sequence analysis; mass spectrometric analysis;SDS-PAGE analysis to compare reduced and intact antibody; peptide map(for example tryptic or LYS-C) analysis; evaluating biological activityor antigen binding function of the antibody; etc.

In one embodiment, the pharmaceutical formulation of the presentinvention is suitable for subcutaneous or intramuscular administration.

“Percent identity” between a query amino acid sequence and a subjectamino acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTP algorithm when a subjectamino acid sequence has 100% query coverage with a query amino acidsequence after a pair-wise BLASTP alignment is performed. Such pair-wiseBLASTP alignments between a query amino acid sequence and a subjectamino acid sequence are performed by using the default settings of theBLASTP algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query amino acid sequence may be described by anamino acid sequence identified in one or more claims herein.

The query sequence may be 100% identical to the subject sequence, or itmay include up to a certain integer number of amino acid alterations ascompared to the subject sequence such that the % identity is less than100%. For example, the query sequence is at least 50, 60, 70, 75, 80,85, 90, 95, 96, 97, 98, or 99%6 identical to the subject sequence. Suchalterations include at least one amino acid deletion, substitution(including conservative and non-conservative substitution), orinsertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the query sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the query sequence or in one or more contiguous groupswithin the query sequence.

The % identity may be determined across the entire length of the querysequence, including the CDR(s). Alternatively, the % identity mayexclude the CDR(s), for example the CDR(s) is 100% identical to thesubject sequence and the % identity variation is in the remainingportion of the query sequence, so that the CDR sequence is fixed/intact.

In one embodiment, the antigen binding protein is a monoclonal antibodyor fragment thereof. In one embodiment, the monoclonal antibody orfragment thereof is mouse, chimeric, humanized, or fully human. In oneembodiment, the monoclonal antibody or fragment thereof binds to BLyS(SEQ ID NO: 1) or a hetero- or homo-trimeric form of BLyS, for example,the monoclonal antibody or fragment thereof binds to the soluble form ofBLyS (SEQ ID NO: 10). In one embodiment, the monoclonal antibodycomprises heavy and light chain variable regions comprising amino acidsequences that are 90% identical to, or is 91% identical to, or is 92%identical to, or is 93% identical to, or is 94% identical to, or is 95%identical to, or is 96% identical to, or is 97% identical to, or is 98%identical to, or is 99% identical to SEQ ID NO: 2 and 90% identical to,or is 91% identical to, or is 92% identical to, or is 93% identical to,or is 94% identical to, or is 95% identical to, or is 96% identical to,or is 97% identical to, or is 98% identical to, or is 99% identical toSEQ ID NO: 3, respectively, or amino acid sequences that are 90%identical to, or is 91% identical to, or is 92% identical to, or is 93%identical to, or is 94% identical to, or is 95% identical to, or is 96%identical to, or is 97% identical to, or is 98% identical to, or is 99%identical to SEQ ID NO: 4 and 90% identical to, or is 91% identical to,or is 92% identical to, or is 93% identical to, or is 94% identical to,or is 95% identical to, or is 96% identical to, or is 97% identical to,or is 98% identical to, or is 99% identical to SEQ ID NO: 5,respectively. In one embodiment, the monoclonal antibody comprises heavyand light chain variable regions comprising amino acid sequences thatare 95% identical to SEQ ID NOs: 2 and 3, respectively, or amino acidsequences that are 95% identical to SEQ ID NOs: 4 and 5, respectively.In one embodiment, the monoclonal antibody comprises heavy and lightchain variable regions comprising amino acid sequences that are 90%identical to SEQ ID NOs: 2 and 3, respectively, or amino acid sequencesthat are 90% identical to SEQ ID NOs: 4 and 5, respectively. In oneembodiment, the monoclonal antibody comprises heavy and light chainvariable regions comprising amino acid sequences set out in SEQ ID NOs:2 and 3, respectively, or SEQ ID NOs: 4 and 5, respectively. In oneembodiment, the monoclonal antibody comprises heavy and light chainscomprising amino acid sequences that are 90% identical to, or is 91%identical to, or is 92% identical to, or is 93% identical to, or is 94%identical to, or is 95% identical to, or is 96% identical to, or is 97%identical to, or is 98% identical to, or is 99%6 identical to SEQ ID NO:6 and 90% identical to, or is 91% identical to, or is 92% identical to,or is 93% identical to, or is 94% identical to, or is 95% identical to,or is 96% identical to, or is 97% identical to, or is 98% identical to,or is 99% identical to SEQ ID NO: 7, respectively, or amino acidsequences that are 90% identical to, or is 91% identical to, or is 92%identical to, or is 93% identical to, or is 94% identical to, or is 95%identical to, or is 96% identical to, or is 97% identical to, or is 98%identical to, or is 99% identical to SEQ ID NO: 8 and 90% identical to,or is 91% identical to, or is 92% identical to, or is 93% identical to,or is 94% identical to, or is 95% identical to, or is 96% identical to,or is 97% identical to, or is 98% identical to, or is 99% identical toSEQ ID NO: 9, respectively. In one embodiment, the monoclonal antibodycomprises heavy and light chains comprising amino acid sequences thatare 95% identical to SEQ ID NOs: 6 and 7, respectively, or amino acidsequences that are 95% identical to SEQ ID NOs: 8 and 9, respectively.

In one embodiment, the monoclonal antibody comprises heavy and lightchains comprising amino acid sequences that are 90% identical to SEQ IDNOs: 6 and 7, respectively, or amino acid sequences that are 90%identical to SEQ ID NOs: 8 and 9, respectively. In one embodiment, themonoclonal antibody comprises heavy and light chains comprising aminoacid sequences set out in SEQ ID NOs: 6 and 7, respectively, or SEQ IDNOs: 8 and 9, respectively. In one embodiment, the monoclonal antibodycomprises CDRs comprising amino acid sequences set out in SEQ ID NOs:11, 12, 13, 14, 15, and 16. In one embodiment, the anti-BLyS antibody isselected from the group of belimumab, tabalumab, and a mixture thereof.In one embodiment the anti-BLyS antibody comprises the heavy and lightchain sequences set out in SEQ ID NOs: 6 and 7, respectively.

In one embodiment, the pharmaceutical formulation according to thepresent invention comprises a monoclonal antibody concentration of200±20 mg/mL. In one embodiment the antibody concentration is about 200mg/mL. In one embodiment the anti-BLyS antibody is co-administeredconcomitantly or sequentially with a corticosteroid. In one embodiment,the corticosteroid is selected from the group consisting of prednisone,prednisolone, hydrocortisone, methylprednisolone and dexamethasone. Inone embodiment, the corticosteroid is prednisone.

In one aspect, the present invention provides for a pharmaceuticalformulation according to any preceding claim for the treatment of adisease or disorder amenable to treatment with an anti-BLyS antibody. Inone embodiment, the present invention is directed to a method oftreating a disease or condition which is amenable to treatment with ananti-BLyS antibody in a subject comprising administering a formulationaccording to the present invention in a subject in an amount effectiveto treat the disease or condition. In one embodiment the disease orcondition is selected from the group consisting of systemic lupuserythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis,lupus nephritis, primary Sjögren's syndrome, chronic immunethrombocytopenia, myasthenia gravis, symptomatic Waldenström'smacroglobulinaemia, immune desensitizing of patients awaiting kidneytransplant, membranous nephropathy, systemic sclerosis, rheumatoidarthritis, multiple myeloma, multiple sclerosis, and kidney failure. Inanother embodiment the disease or condition is systemic lupuserythematosus. In another aspect the present invention provides for aformulation for use in the treatment of disease selected from the groupconsisting of systemic lupus erythematosus, anti-neutrophil cytoplasmicantibody (“ANCA”) vasculitis, lupus nephritis, primary Sjögren'ssyndrome, chronic immune thrombocytopenia, myasthenia gravis,symptomatic Waldenström's macroglobulinaemia, immune desensitizing ofpatients awaiting kidney transplant, membranous nephropathy, systemicsclerosis, rheumatoid arthritis, multiple myeloma, multiple sclerosis,and kidney failure. In another aspect the present invention provides fora formulation for use in the treatment of systemic lupus erythematosus.In another aspect the present invention provides for the use of aformulation in the preparation of a medicament for the treatment of adisease selected from the group consisting of systemic lupuserythematosus, anti-neutrophil cytoplasmic antibody (“ANCA”) vasculitis,lupus nephritis, primary Sjögren's syndrome, chronic immunethrombocytopenia, myasthenia gravis, symptomatic Waldenström'smacroglobulinaemia, immune desensitizing of patients awaiting kidneytransplant, membranous nephropathy, systemic sclerosis, rheumatoidarthritis, multiple myeloma, multiple sclerosis, and kidney failure. Inanother aspect the present invention provides for the use of aformulation in the preparation of a medicament for the treatment ofsystemic lupus erythematosus.

In one aspect, the present invention provides for a kit comprising oneor more vials containing the formulation of the present invention andinstructions for subcutaneous administration of the formulation to apatient. In one embodiment, the kit further comprises an injectiondevice for subcutaneous administration of the formulation to a patient.

In one embodiment, the present invention is directed to an injectiondevice comprising a stable anti-BLyS antibody formulation describedherein. For subcutaneous delivery, the formulation may be administeredvia a suitable device, such as (but not limited to) a syringe; aninjection device (e.g. the INJECT-EASE™ and GENJECT™ device); aninfusion pump (such as e.g. Accu-Chek™); an injector pen (such as theGENPEN™; or a needleless device (e.g. MEDDECTOR™ and BIOJECTOR™).

The pharmaceutical formulation in accordance with the invention isessentially free from visible (human eye inspection) particles. Thesub-visible particles (as measured by light obscuration) should fulfillthe following criteria: maximum number of particles ≧10 μm pervial→6000; maximum number of particles ≧25 μm per vial→600.

The pharmaceutical formulation of the pharmaceutically active anti-BLySantibody in accordance with the invention can be administered assubcutaneous injection, whereby the administration is repeated severaltimes with time intervals of 1, 2, 3, or 4 weeks. In one embodiment thepharmaceutical formulation of the pharmaceutically active anti-BLySantibody is administered once every week or once every two weeks. Thefull volume of the injection fluid is in most cases administered withina time period of 1 to 10 minutes, preferably 2 to 6 minutes, mostpreferably 3±1 minutes.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes, on theprevious therapy, the patient's clinical history and his response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 50 mg/kg of bodyweight or more specifically between about 0.1mg/kg to 20 mg/kg of bodyweight) of the antibody is a candidate initialdosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. Morespecifically the dosage of the antibody will be in the range from about0.05 mg antibody/kg of bodyweight to about 10 mg antibody/kg ofbodyweight.

In another embodiment of the invention, an article of manufacture isprovided which contains the pharmaceutical formulation of the presentinvention and provides instructions for its use. This article ofmanufacture comprises a container. Suitable containers include, forexample, bottles, vials (e.g. multiple or dual chamber vials), syringes(such as multiple or dual chamber syringes) and test tubes. Thecontainer may be formed from a variety of materials such as glass orplastic. The container holds the formulation and the label on, orassociated with, the container may indicate directions for use. Thecontainer holding the formulation may be a multi-use vial, which allowsfor repeat administrations (e.g. from 2 to 6 administrations) of thereconstituted formulation. The article of manufacture may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

The antibody which is formulated in accordance with the presentinvention is preferably essentially pure and desirably essentiallyhomogeneous. An “essentially pure” antibody means a compositioncomprising at least about 90% by weight of the antibody, based on totalweight of the composition, preferably at least about 95% by weight. An“essentially homogeneous” antibody means a composition comprising atleast about 99% by weight of antibody, based on total weight of thecomposition.

The invention will be more fully understood by reference to thefollowing Examples. They are merely illustrative and should not beconstrued as limiting the scope of the invention. Minor variations inprocedure, e.g. minor changes in time, temperature, quantity,concentration, scale, etc., are not anticipated to affect the outcome ofthe experiments. All literature and patent citations are incorporatedherein by reference.

The Examples are further illustrated by the appended FIGS. 1-20.

Examples Example 1: Belimumab Formulations Container Closure

Schott type I vials with Daikyo D21-7S Flurotec® stoppers and flip offaluminum seals were used in all studies unless otherwise mentioned. Thisvial and stopper combination is recommended as the Phase 1configuration. Long term stability samples that were stored >2-8° C.used Gerresheimer 1.0 mL long, 29 G, thin wall, staked, pre-filledsyringes with Stelmi 4800 needle shields and Daikyo W4023 Flurotec®plungers, and were set by vacuum with a nitrogen overlay. Samples <2-8°C. were filled into cryogenic vials.

Product Handling Procedures

Prior to all experiments, belimumab was sterile filtered with a 0.22 μmfilter and aseptically filled into the chosen container closures. Allstability samples were protected from light during storage.

Excipient Selection

Multi-compendial excipients, mandatory for GMP BDS and FDPmanufacturing, were used where possible in the screening studies, andused for all formulations in the long term stability study.

Table 2 provides a list of the formulations tested.

TABLE 2 Formulation Description Stabilizing Buffer conc excipient concIdentifier (mM) (mM) pH 1 10 mM Histidine 140 mM NaCl 6.0 2 10 mMHistidine 280 mM Sucrose 6.0 3 10 mM Histidine 140 mM Sucrose, 6.0 70 mMNaCl 4 10 mM Histidine 5.4 mM MgCl₂, 6.0 130 mM NaCl 5 10 mM Histidine25 mM Arg, 6.0 115 mM NaCl 6 10 mM Histidine 280 mM Sorbitol 6.0 7 10 mMHistidine 25 mM Arg, 6.0 5.4 mM MgCl₂, 105 mM NaCl 8 10 mM Succinate 140mM NaCl 6.0

Long Term Stability Concentration-Dependent Aggregation in Formulation 1

As expected, aggregation increased with protein concentration (Table 3,FIG. 1). The aggregation rate approximately doubles between 100 mg/mLand 260 mg/mL, but even at 260 mg/mL would only lead to approximately 1%increase in aggregation over 3 years at 2-8° C. at 200 mg/mL belimumab.Note the starting amount of aggregation observed by SEC-HPLC increasesas protein concentration increases, although only by approximately 0.1%(0 month row of Table 3).

TABLE 3 Effect of Protein Concentration on % Aggregate 100 140 180 220260 Month mg/mL mg/mL mg/mL mg/mL mg/mL 0 0.47% 0.50% 0.54% 0.58% 0.60%1 0.47% 0.51% 0.52% 0.58% 0.62% 2 0.54% 0.61% 0.64% 0.75% 0.78% 4 0.53%0.59% 0.63% 0.72% 0.77% 6 0.58% 0.63% 0.70% 0.76% 0.84% Rate/Month0.018% 0.021% 0.027% 0.030% 0.040%

Long Term Formulation Candidate Screen

Based on the following results, the formulation candidates were narrowedto the Formulations 1 and 5 after evaluation of the 3 month data, andthen Formulation 5 was chosen as the final formulation after 5¼ months.

Appearance, pH, and Osmolality

All samples were opalescent, pale yellow, and free from visibleparticulate matter at all time points up to 5¼ months. Finished drugproduct in all eight formulations at all three concentrations mostclosely matched the Y5 color standard when tested by colorimetry at theinitial time point. All FDP samples in the histidine/NaCl (hereinreferred to as Formulation 1) and the histidine/NaCl/arginine (hereinreferred to as Formulation 5) formulations also matched the Y5 standardafter 3 and 5′ months of storage at 2-8° C. Turbidity of the sampleswith sugar stabilizers (sucrose and sorbitol) was significantly lowerthan all other samples, which ranged from 29-38 NTU at the initial and 3month time point. Turbidity also increased in the NaCl containingsamples as the protein concentration decreased. Only the Formulations 1and 5 were tested after 5¼ months at 2-8° C., and showed no response toformulation, concentration or time (FIG. 2).

TABLE 4 Turbidity of Long Term Stability Samples after 3 months Storageat 2-8° C. Turbidity (NTU) High Mid Low Concen- Concen- Concen- trationtration tration (~200 (~165 (~125 mg/mL) mg/mL) mg/mL) 0 3 0 3 0 3 MonthMonth Month Month Month Month Hist/NaCl 33 33 37 37 37 37(Formulation 1) Hist/sucrose (2) 25 23 24 24 22 23 Hist/sucrose/ 30 3033 33 34 35 NaCl (3) Hist/NaCl/ 31 31 35 36 36 37 MgCl₂ (4)Hist/NaCl/Arg 29 28 33 33 33 34 (Formulation 5) Hist/ 27 27 24 26 26 26Sorbitol (6) Hist/NaCl/Arg/ 29 29 32 31 32 33 MgCl₂ (7) Succinate/ 31 3136 35 36 37 NaCl (8)

The pH of all samples ranged from 6.1 to 6.3 at the initial time point,and did not shift in Formulations 1 and 5 after 5¼ months (Formulation 5data shown in Table 13). Osmolality was tested only at the initial timepoint; all samples were 299+/−17 mOsm/kg.

Viscosity and Syringability

The sugar containing formulations (sucrose, sorbitol) showed the highestviscosities, followed by the succinate/sodium chloride formulation(Table 5). The rest of the salt containing samples were comparable.Viscosity increased exponentially as protein concentration increased inFormulations 1 and 5 (FIG. 3).

TABLE 5 Viscosity of Long Term Stability Samples at T0 Viscosity (mm2/s)High Mid Low Concen- Concen- Concen- tration tration tration (~200 (~165(~125 Sample mg/mL) mg/mL) mg/mL) Hist/NaCl 10.2 5.8 3.6 (Formulation 1)Hist/sucrose 17.4 7.7 4.9 Hist/sucrose/NaCl 13.4 7.9 3.9 Hist/Nacl/MgCl₂12.8 5.6 3.4 Hist/Nacl/Arg 13.0 5.8 3.8 (Formulation 5) Hist/Sorbitol21.0 7.6 4.3 Hist/NaCl/Arg/MgCl₂ 12.8 5.6 3.5 Succinate/NaCl 15.3 6.63.8

Syringability, measured as force required to deliver 1 mL through thethin walled 29 G needle in 10 seconds, showed similar trends at theinitial time point. After 5¼ months, only Formulations 1 and 5 weretested, and there was no significant increase in syringability observedover time at 2-8° C. Syringability over 20 seconds was also tested onone of each syringe at the 5 month time point, and delivery force wasshown to decrease by up to 40% when delivery time doubled. While nottested, delivery force can also be decreased by increasing needle gauge.

TABLE 6 Syringability of Long Term Stability Samples at T0 and 5¼ MonthsSyringability (N) High Mid Low Concen- Concen- Concen- tration trationtration Sample and (~200 (~165 (~125 Delivery Time Sample mg/mL) mg/mL)mg/mL) Initial Time point Hist/NaCl 21.9 14.5 12.0 10 Second(Formulation 1) Delivery Hist/sucrose 31.3 18.2 13.8 Hist/sucrose/NaCl25.7 17.5 12.7 Hist/NaCl/MgCl₂ 23.8 14.1 11.1 Hist/NaCl/Arg 24.4 16.311.5 (Formulation 5) Hist/Sorbitol 35.7 18.0 12.6 Hist/NaCl/Arg/MgCl₂25.0 16.1 10.9 Succinate/NaCl 28.3 14.6 11.7 5¼ month, Hist/NaCl 24.416.1 10.6 2-8° C. Sample (Formulation 1) 10 Second Hist/Nacl/Arg 24.216.7 10.8 Delivery (Formulation 5) 5¼ month, Hist/NaCl 14.4 10.0 9.42-8° C. Sample (Formulation 1) 20 Second Hist/Nacl/Arg 16.2 11.1 6.4Delivery (Formulation 5)

Forces required to administer drug through seven marketed pen injectors,which are more similar to pre-filled syringes because they require amanual driving force, are similar to the force for belimumab at 200mg/mL (Table 7). The injection times varied because of different volumesand container diameters, which are listed in Table 7 for comparison.Finally, a University of Nottingham study commissioned by the UK'sDepartment of Trade and Industry has shown that while seated, 59 womenbetween the ages of 16 and 90 were able to apply 53.7 to 237.7N ofdownward static force at hip level with their thumb. Although neitherthe pen injector data nor the force study are perfect correlators forusing a pre-filled syringe, both data sets build confidence that theviscosity and syringability of 200 mg/mL belimumab are not prohibitivefor manual administration. However, the force required to deliver 200mg/mL belimumab from a 1 mL long pre-filled syringe through a 29 G thinwall needle is at or near the desirable limit for manual injection, anda wider needle would be preferred.

TABLE 7 Injection Forces of Commercially Available Pens at 80 mm/minTime Injec- to tion Deliver Provided Force (sec- Pen Needle CompanyIndication (N) onds) Gonal-f Ypsomed EMD Serono Infertility 22.3 5.3 RFFPenfine 29 G × ½″ Lantus None (BD Sanofi-Aventis type 2 or 6.4* 11.3SoloStar recommended) Type I diabetes Lantus BD Microfine Scheringchronic 21.1 7.1 SoloStar 30 G × 8 mm Corporation hepatitis C LantusNone (BD Eli Lilly diabetes 24.9* 7.1 SoloStar recommended) Lantus None(29, 30, Amylin type 2 13.4* 4.1 SoloStar 31 G Pharma- diabetesrecommended) ceuticals Eli Lilly Lantus None Novo Nordisk GH 13.8* 9.4SoloStar (Novofine treatment recommended) Lantus None (BD Eli Lillyosteo- 24.1* 5.3 SoloStar recommended) porosis *Delivered using aYpsomed Penfine 29 G × 12.7 mm needle

Size Variants 3 Month SEC-HPLC Data

Aggregation, seen by SEC-HPLC, was the predominateconcentration-dependent pathway for belimumab in all formulations.Percent fragmentation (observed as a back shoulder) was variable between0.1 and 0.2%, but did not change over time (supported by 5¼ month data).

After 3 months at 2-8° C., distinct differences in aggregation rate(FIG. 4) were observed among belimumab formulated in the eightformulations. Formulation 5 (histidine/NaCl/arginine) showed the lowestrate over three months, particularly at 200 mg/mL (blue in FIG. 4). Thiswas supported by the accelerated trends at 200 mg/mL (FIG. 5). Succinatewas the worst stabilizer at low temperatures, but the best at elevatedtemperatures. Many of the other salt and sugar formulations, includingFormulation 1 (histidine/NaCl), showed similar absolute aggregatepercentages and aggregation rates.

5¼ Month SEC-HPLC Data

Belimumab in Formulations 1 and 5 were evaluated at 5 months. The trendsobserved at 3 months continued, with the arginine containing formulationshowing a lower aggregation rate, especially at the highestconcentration of 200 mg/mL. FIG. 6 shows aggregation rates after 5months at up to 25° C., and shows that the arginine formulation (opensquares on the graph) significantly dampens aggregation when compared toFormulation 1 (filled squares). Further analysis of the aggregationrates at various temperatures in FIG. 7 shows how consistentlyFormulation 5 (dashed lines) shows a lower aggregation rate thanFormulation 1 (solid lines). If the 2-8° C. aggregation rate observed upto 5¼ months holds through 3 years, FDP would only increase byapproximately 1.2%.

CGE

Reducing capillary gel electrophoresis of Formulations 1 and 5 showed notrends after 5 months of storage at various temperatures (rates shown inFIG. 8). Cross-linking and clipping are therefore not dependent onconcentration or formulation.

Charge Heterogeneity

Ion exchange indicates neither concentration nor the addition ofarginine to a histidine buffered salt formulation impacts chargevariants (FIG. 9). Although acidic variants increase over time atelevated temperatures, little to no change in variants was observedafter 5 months.

Oxidation

No significant changes in oxidation were observed among any of the 8formulations after 3 months storage at 2-8° C. (data not shown). After 5months, when comparing −80° C. and 15° C. data, no differences inoxidation were observed between Formulations 1 and 5 or among the threeconcentrations in either formulation (FIG. 10). Approximately 1.0%additional oxidation was observed in all samples after 5¼ months storageat 25° C., and approximately 4.5% additional oxidation was observed at40° C.

Peptide Mapping

No differences were observed among the 200 mg/mL Formulation 1 −80° C.and 2-8° C. samples or the reference standard after 5′/% months (FIG.11). The 25° C. sample showed a small increase in T4 deamidation, asexpected at accelerated temperatures. The Formulation 5 sample similarlyshowed T4 deamidation only at 25° C., but also showed inconsistent peakheights in a number of other peptide peaks (T33, T34 and T5 of the heavychain, T3 of the light chain in FIG. 12). These peak heights did nottrend with temperature, so digestion interference by arginine wassuspected.

To determine if arginine interference was the cause of the variability,0, 25 and 50 mM arginine were added to a Formulation 1 sample which hadbeen through the method's desalting step. All three samples were thenrun through the remaining steps, which include trypsin digestion. Thesame peptide peaks that showed variability in the stability samplesshowed responses that correlated with arginine concentration (FIG. 13).This indicates the arginine may not always be fully cleared by thedesalting step, and explains the temperature independent variationobserved in the peptide maps of samples formulated in Formulation 5.Because no other modifications in the peptide maps were observed, it canbe surmised that despite the differences in the maps, the Formulations 1and 5 had no observable degradation after 5¼ months at 2-8° C., and onlyminimal degradation after 5¼ months at 25° C.

Potency

Belimumab remains biologically active after storage at 2-8° C. for 3months in either Formulation 1 or Formulation 5 between 125 and 200mg/mL, or after 5¼ months in Formulation 5 at 200 mg/mL (Table 8).

TABLE 8 Relative Potency after Stability at 2-8° C. % Relative Potency 3Months 5¼ Months 200 mg/mL Formulation 5 102% 118% 125 mg/mL Formulation5 106% 200 mg/mL Formulation 1  90% 125 mg/mL Formulation 1 102%

Evaluation of Freeze/Thaw

Samples exposed to 5 fast freeze/thaw cycles between −40° C. and 2-8° C.had a similar amount of aggregation as the −40° C. control samples,indicating fast freeze/thaws are not a concern in either Formulation 1or the Formulation 5 (Table 9).

TABLE 9 SEC-HPLC Results from belimumab Exposed to Fast Freeze/Thaw %Aggregate Formulation 1 Formulation 5  2-8° C. ctrl 0.6 0.6 −20° C. ctrl0.8 0.8 −40° C. ctrl 0.6 0.7 −80° C. control 0.6 0.6 −40/2-8° C. 5xCycle 0.7 0.7

Samples exposed to 3 slow freeze/thaw cycles showed a 0.2% increase inaggregate level compared to liquid controls (Table 10).

TABLE 10 SEC-HPLC Results from belimumab Exposed to Slow Freeze/ThawSample % Aggregate % Main Peak % Clip Formulation 1 Liquid Control 0.799.1 0.2 Formulation 1 Slow freeze/thaw 0.9 98.9 0.2 Formulation 5Liquid Control 0.6 99.2 0.1 Formulation 5 Slow freeze/thaw 0.8 99.1 0.2

DSC

Calorimetry was used to assess the sub-freezing glass transition (Tg′)of each formulation and to determine whether a sub-freezing eutectic wasformed. Sodium chloride—water eutectic can form below approximately −21°C. and eutectic crystallization of excipients may affect product qualityby introducing crystalline surface interactions and changing the localchemical environment in the freeze concentrate containing protein.Storage below Tg′ may improve stability by increasing relaxation timeand reducing associated degradation.

Formulations 1 and 5 of high concentration belimumab had similarbehavior with respect to sub-freezing transitions (Table 11). ForFormulation 1, Tg′ ranged from −23° C. (fastest freeze) to −33° C.(slowest freeze). For Formulation 5, Tg′ ranged from −22° C. (fastestfreeze) to −32° C. (slowest freeze). For both formulations, a eutecticendotherm was observed only after thermal cycling with multipleannealing steps at −23° C. The eutectic was most likely sodiumchloride—water.

These results indicate that the sub-freezing thermal transitions ofthese formulations are sensitive to the thermal history of the sample.This is likely due to the high dissolved solids content and the presenceof sodium chloride, which can affect Tg′ in the protein/amorphous phase.The results, in conjunction with the −80° C. and −40° C. stability datafrom Section 5.2, also indicate the BDS storage <−40° C. and protectedfrom light is sufficient for belimumab in Formulation 5.

TABLE 11 DSC Results for belimumab in Formulation 5 & Formulation 1Sample Formulation 1: Formulation 5: Fast Freeze Sample mass 16.5 16.5(mg) Tg′ −23° C. −22° C. Eutectic No eutectic No eutectic endothermobserved observed Medium freeze Sample mass 15.7 16.7 to −40° C. (mg)anneal Tg′ −27° C. −26° C. Eutectic No eutectic No eutectic endothermobserved observed Medium freeze Sample mass 16.9 16.3 to −23° C. (mg)anneal Tg′ −25° C. −27° C. Eutectic No eutectic No eutectic endothermobserved observed Slow freeze Sample mass 17.7 16.3 to −60° C. (mg) Tg′Weak −33° C. Weak −32° C. Eutectic No eutectic No eutectic endothermobserved observed Thermal Sample mass 15.3 18.6 cycling (mg) (−80/−23°C. Tg′ After cycling, After cycling, with annealing) weak −31° C. weak−28° C. and −18° C. Eutectic Between −16° C. Between −16° C. endothermand −12° C. and −12° C. (0.6 J/g) (0.7 J/g)

Evaluation of Shaking

After 48 hours of shaking at 250 rpm, there was no significant change inpurity by SEC-HPLC or turbidity in either the vial or the syringe overthe range of polysorbate concentrations studied (Table 12). 0.01%polysorbate 80 was shown to be effective and robust in Formulation 5 asa protectant against shaking in both a vial and a syringe.

TABLE 12 SEC-HPLC and Turbidity of belimumab in Formulation 5 afterShaking at 250 rpm Container % Main Peak by SEC Turbidity (NTU) ClosureSample 0 hr. 24 hr. 48 hr. 0 hr. 48 hr. Vial Control (No 99.4 99.3 99.337 38 Shaking) Low PS80 99.4 99.3 99.3 35 35 (0.005%) Target PS80 99.499.3 99.3 33 37 (0.01%) High PS80 99.4 99.4 99.4 30 33 (0.02%) SyringeControl (No 99.4 99.3 99.3 37 38 Shaking) Low PS80 99.4 99.3 99.3 35 35(0.005%) Target PS80 99.4 99.3 99.3 33 34 (0.01%) High PS80 99.4 99.399.4 30 32 (0.02%)

CONCLUSIONS

A formulation for subcutaneous administration of belimumab at 200 mg/mLwas chosen based on its ability to minimize the primary degradationpathway rates (Formulation 5; 0.65 mg/mL L-histidine, 1.2 mg/mLL-histidine monohydrochloride, 6.7-7.3 mg/mL sodium chloride, 5.3 mg/mLL-arginine hydrochloride, 0.1 mg/mL polysorbate 80, pH 6.0; or,alternatively, 10 mM histidine, 115 mM sodium chloride, 25 mM L-argininehydrochloride, 0.01% (w/v) polysorbate 80, pH 6.0). The aggregation rate(˜0.03%/month at 2-8° C.) was shown to increase with belimumabconcentration, but was inhibited by the use of 25 mM arginine. Thedeamidation rate was approximately 0.2%/month at 2-8° C. The 200 mg/mLformulation has an acceptable delivery force for manual or autoinjectordelivery using a 1 mL long syringe and a 29 G thin wall or wider needle.Freeze/thaw profiles and storage at −80° C. and −40° C. were shown to beacceptable, and the product is not susceptible to shaking stress.

Long term GMP stability studies were performed on 200 mg/mL belimumabfinal drug product in Formulation 5 (1.0 mL filled in a 1.0 mL long BDsyringe). To date there is 42 months of GMP stability data at intendedstorage temperature of 2-8° C. (Table 14). The results indicate thatFormulation 5 provides adequate stability to belimumab with acceptabledegradation profiles observed at the intended storage temperature of2-8° C. (Table 14).

TABLE 13 Long Term Stability of belimumab in Formulation 5 at 200 mg/mLSEC SEC IEC RP % Potency Turbid- % SEC % IEC IEC IEC Basic HC CGE (%Time Temp Appear- Color- ity Aggre- % Frag- % % Basic (w/o Oxida- %Relative (Month) (° C.) ance imetry pH (NTU) gate Purity ment AcidicMain Shoulder shoulder) tion Purity Binding) 0 NA OPF Y5 6.3 29 0.6 99.20.2 12.9 78.5 5.2 8.6 1.8 95.0 3 −80 6.2 0.6 99.4 0 12.6 79.4 5.0 8.03.8 93.1 102 −40 6.2 0.6 99.3 0.1 12.7 79.6 4.8 7.7 3.8 93.8 5 OPF Y56.3 29 0.7 99.1 0.2 13.0 79.3 4.9 7.7 2.2 92.4 105 15 Y5 6.3 1.0 98.80.2 15.6 76.9 4.6 7.5 4.1 95.7 25 Y5 6.1 1.2 98.5 0.4 25.7 68.1 3.9 6.24.6 93.2 5.25 −80 6.2 0.6 99.4 0.0 13.9 78.2 4.7 7.9 2.1 94.7 −40 6.20.7 99.3 0.0 12.2 78.9 5.6 9.0 2.0 95.6 5 OPF Y5 6.2 37 0.7 99.2 0.112.9 78.2 5.1 8.8 2.3 94.3 118 15 6.2 1.0 98.8 0.1 18.1 73.1 5.5 8.8 2.492.8 25 6.2 1.4 98.2 0.4 34.2 58.5 4.1 7.3 3.3 93.1 40 6.2 9.7 88.1 2.292.9 2.2 3.3 4.8 6.4 83.7 9 5 OPF Y5 0.9 99.1 0.1 14.6 78.3 4.2 7.2 2.696.4

TABLE 14 GMP Stability Data of 200 mg/mL Belitnumab in Formulation 5 atIntended Storage Temperature of 2-8° C. (1.0 mL in a 1.0 mL Long BDSyringe) Test (Analytical Clinical Acceptance Time (Months) Method)Criteria 0 1 2 3 6 9 12 18 24 30 36 42 Appearance Clear to opalescent,OPF OPF OPF OPF OPF OPF OPF OPF OPF OPF OPF OPF [Visual colorless topale Inspection yellow solution, (Ph. Eur. essentially free from 2.2.1and foreign particulate 2.2.2)] matter Charge Main Peak (MP): MP: 75.6MP: 75.3 MP: 74.9 MP: 74.7 MP: 75.0 MP: 74.1 MP: 72.0 MP: 72.3 MP: 70.7MP: 69.4 MP: 69.2 MP: 68.3 Heterogeneity 64.3-89.7% (IE-HPLC) AcidicPeaks (AP): AP: 13.2 AP: 13.4 AP: 13.6 AP: 14.1 AP: 14.1 AP: 14.9 AP:16.3 AP: 16.2 AP: 17.7 AP: 19.3 AP: 20.1 AP: 20.8 Report result (X.X %)Container Pass Pass Pass Pass Pass Pass Closure Integrity (Dye leak)Deliverable 1.0-1.1 mL Max: 1.06 Max: 1.06 Max: 1.06 Max: 1.06 Max: 1.08Max: 1.07 Max: 1.06 Max: 1.06 Max: 1.06 Max: 1.06 Max: 1.06 Max: 1.07Volume (USP <1>, Maximum volume: Ph. Eur. X.XX mL 2.9.17) Minimumvolume: Min: 1.04 Min: 1.05 Min: 1.05 Min: 1.05 Min: 1.05 Min: 1.05 Min:1.05 Min: 1.05 Min: 1.05 Min: 1.05 Min: 1.04 Min: 1.05 X.XX mL InjectionMaximum Break BLF: 7.7 BLF: 7.0 BLF: 7.8 BLF: 7.1 BLF: 7.5 BLF: 7.1 BLF:8.0 BLF: 8.1 BLF: 7.3 BLF: 6.8 BLF: 8.4 BLF: 9.5 Forces Loose Force:(Compression Report result Force Test (X.X N) Stand) Maximum Peak PEF:7.4 PEF: 9.1 PEF: 8.1 PEF: 8.6 PEF: 7.5 PEF: 7.2 PEF: 7.6 PEF: 9.2 PEF:9.3 PEF: 7.5 PEF: 9.9 PEF: 8.4 Extrusion Force: Report result (X.X N) pH[pH 6.0 +/− 0.4 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.1 6.0 6.0 6.1 6.1Electrode (USP <791>, Ph. Eur. 2.2.3)] Potency 75-133% relative 105 10596 98 98 99 108 93 105 96 98 98 (Inhibition of potency Binding) Protein200 +/− 40 mg/mL 200 204 195 202 197 200 198 197 199 197 198 197Concentration (A280) Purity (SDS- >=95% 99 98 99 98 99 98 99 99 99 99 9999 PAGE: Reduced with Coomassie Blue Stain) Purity (SEC- Main Peak MP:99.4 MP: 99.4 MP: 99.2 MP: 99.2 MP: 99.2 MP: 99.1 MP: 99.1 MP: 99.0 MP:98.9 MP: 98.8 MP: 98.7 MP: 98.8 HPLC) (MP): >=95.0% Aggregate (AG): AG:0.5 AG: 0.6 AG: 0.7 AG: 0.7 AG: 0.8 AG: 0.8 AG: 0.8 AG: 0.9 AG: 1.0 AG:1.0 AG: 1.1 AG: 1.0 Report result (X.X %) Fragment (FG): FG: 0.1 FG: 0.0FG: 0.0 FG: 0.0 FG: 0.1 FG: 0.1 FG: 0.1 FG: 0.1 FG: 0.1 FG: 0.1 FG: 0.2FG: 0.1 Report result (X.X %) Sterility No growth No growth (USP <71>,Ph. Eur. 2.6.1) Subvisible Meets Meets Meets Meets Meets Meets MeetsParticulate USP <788> USP <788> USP <788> USP <788> USP <788> USP <788>USP <788> Matter and Ph. and Ph. and Ph. and Ph. and Ph. and Ph. and Ph.(USP <788>, Eur. 2.9.19 Eur. 2.9.19 Eur. 2.9.19 Eur. 2.9.19: Eur. 2.8.19Eur. 2.9.19 Eur. 2.9.19 Ph. Eur. <=6000 particles per 62 45 290 176 179293 2.9.19) container >=10 um <=600 particles per 6 0 12 16 7 10container >=25 um Appearance key: C = Clear, O = Opalescent; L =Colorless, P = Pale yellow, Y = Yellow; F = essentially free fromforeign particulate matter; X = other Injection Force: BLF = Break LooseForce, PEF = Peak Extrusion Force,

Example 2: Anti-IL13 is a High Concentration-High Dose mAb

Anti-IL13 is a glycosylated humanized mAb (IgG1) directed against humaninterleukin-13 (IL13). In order to achieve a very high clinical dose (10mg/kg) for subcutaneous delivery, based on PK/PD modelling, the drugsubstance and drug product needed to be developed at a concentration of200 mg/mL in a vial presentation. As a direct consequence of achieving amuch higher concentration of mAb in the vial, various formulationchallenges for anti-IL13 were presented such as: (i) to identify aunique formulation able to support the stability, manufacturability,analytical and delivery challenges of a high concentration monoclonalantibody intended for delivery at a high clinical dose via subcutaneousdelivery, (ii) to prevent analytical and stability challenges arisingdue to gelation of the monoclonal antibody at higher concentrations(iii) to prevent various related challenges that may arise especiallyduring delivery of the monoclonal antibody in an injection volume of 1.5mL or less. As a direct finding revealed during the formulationdevelopment work, it was determined that this monoclonal antibody had atendency to form irreversible gel like matrix in certain buffer systemsat elevated temperatures, therefore, posing significant risk ofinstability of the protein. Viscosity increases exponentially withconcentration and may complicate the filtration process. Higherconcentration monoclonal antibodies have greater susceptibility toaggregation with increased risks of particulates formation andreversible self association. Therefore, high-throughput formulation(HTF) development studies were performed to identify the optimal bufferand pH for the new formulation to support high concentration monoclonalantibody designed to provide a high clinical dose. These along withother bench top studies identified an optimal formulation whichprevented the gelation phenomenon at elevated temperatures. Theadditional formulation development studies were identified variousexcipients to be included to selected buffer system. These were shake,freeze-thaw, and elevated temperatures studies to assess the physicalstability followed by short and long-term stability studies to assessthe chemical stability of the anti-IL13 mAb. Various other studies werealso conducted to ensure that the clinical delivery considerations aremet.

Example 3: HTF pH-Buffer Screening: (Identification of Target pH andBuffer)

Previously tested acetate based formulation for anti-IL13 monoclonalantibody at 50 mg/mL had already established that the pH was notoptimal. Suboptimal formulation buffer pH could increase instability ofa higher concentration anti-IL13 monoclonal antibody solution byaltering the charge on the protein and affecting electrostaticinteractions. The high concentration formulation development commencedwith the identification of an optimum pH as well as determination of thebest buffer species.

The study was performed at a mAb concentration of 13 mg/mL. The bufferscreening was conducted by means of a HTF process in 96 well plates. Thestudy consisted of a vast range of buffer types and pH levels. Eachplate consisted of 48 samples with 2 replicates in random order. Sampleswere stressed for 3 days at 50° C./ambient RH. The testing includedgeneral appearance (GA), concentration at A280 and A260 nm, pH, sizeexclusion chromatography (SEC), capillary iso-electric focusing (cIEF)and dynamic light scattering (DLS). The tested factors are presented inTable 15.

TABLE 15 HTF Buffer Screen Study Factor 1 Factor 2 Buffer Type pH (3levels) 10, 55, and 100 mM (6 levels) Acetate 4 Acetate, Citrate 5Acetate, Citrate, Succinate 5.5 Acetate, Citrate, Histidine, Succinate 6Acetate, Citrate, Histidine, Succinate 6.5 Histidine, Phosphate 7

The result of the general appearance testing of the plates revealed highlevels of precipitation in some of the samples especially in Acetate,Citrate and Phosphate buffer. Succinate buffer was the only bufferspecies which did not indicate precipitation.

Deamidation and aggregation profiles were generated for all the samplesvia cIEF and SEC, respectively. FIG. 14 shows a plot with the overlay ofcIEF and SEC data. The plot displayed that the formulations with thehighest % Monomer by SEC also had lowered % Main by cIEF indicating thatanti-IL13 aggregates at lower pH and deamidates at higher pH1. It can beobserved that % Monomer is higher where the pH is 6-7 than at pH 4-5.5regardless of buffer species.

Overall, the HTF pH-buffer screening led to the following conclusions:(i) Citrate and acetate buffer resulted in the highest numbers of cloudywells (indication of precipitation) as determined by GA and (ii)Phosphate buffer promoted precipitation along with increase inaggregation and deamidation based on cIEF and SEC results and (iii)anti-IL13 aggregates at lower pH and deamidates at higher pH.

Example 4: HTF pH-Buffer Screening: (Determination of Optimal pH)

A second HTF study was performed based on 3×3 factorial (3 buffers, 3pHs) DOE design consisting of a total of 90 samples. The selectedbuffers for the evaluation included acetate, histidine and succinate atthe final pH ranges between 5.5 and 6.5. The buffers were selected to bea fixed concentration of 25 mM. The design enabled 6 replicates for eachformulation along with 6 replicates of acetate formulation on the stressplate to serve as a control for comparison purposes.

Each plate was stressed for 3 days at 50° C./ambient RH. The testingincluded general appearance (GA), concentration at A280 and A260 nm,pH1. SEC, cIEF, DLS and DSC (Differential Scanning Calorimetry) onselected samples.

The results of the study were analyzed by statistical software known asDesign Expert. Interesting trends were revealed when all the assayresults were subjected to a Analysis of variance (ANOVA) test using thesame software. Buffer species and pH were found to be significantfactors for results by concentration, corrected concentration, DLS, SECand cIEF.

FIG. 15 shows the interaction between pH and buffer species on SEC %monomer. For both acetate and histidine buffers, % monomer increased aspH increased. FIG. 16 shows the interaction of pH11 and buffer on % Mainfor cIEF. Both pH and buffer were found to be significant. From thisplot pH in the range 5.5-6.5 does not appear to affect % Main for eitheracetate or succinate, but it does for histidine.

Overall, the HTF pH-buffer screening led to the selection of finaloptimal pH of 6.25.

Example 5: Determination of an Optimal Thermal Stress Condition Based onThermal Stability Profile by DSC

Among various factors that may potentially influence gelation of proteinsuch as concentration, pH, salt content, there is one critical factorthat governs this phenomenon-temperature. The thermal stabilitycondition selected for anti-IL13 tested by HTF and other developmentstudies was evaluated by DSC. In general, medium sized globular proteinsbegin to unfold around 25° C. and for monoclonal antibodies it is around60° C. The start of unfolding for this mAb under all tested buffers(acetate, histidine and succinate) is around 61° C., indicating athermally-stable molecule, and confirming that a 50° C. acceleratedstorage temperature would allow for a folded anti-IL13 monoclonalantibody. Since there is a difference of more than 10° C. between thestart of unfolding and the 50° C. accelerated storage condition, it wasdetermined to utilize 50° C. as the storage temperature for screening.

Table 16 lists the Tm values as determined from the raw scans. The Tm1values span the 71.3-71.6 C range and the Tm2 values span a larger,83.5-84.1 C, range. There are no significant changes in the Tms of samebuffer at different pH values. The variations in Tm1 and Tm2 as measuredfor all conditions are below 1° C., thus the tested protein solutionshave similar thermodynamic stabilities.

TABLE 16 Tm values as determined from the raw scans Buffer Tm1, C. Tm2,C. acetate 6.0 71.3 84.1 acetate 6.5 71.4 83.9 histidine 6.0 69.9 83.6histidine 6.5 71.3 84.1 succinate 6.0 71.4 83.6 succinate 6.5 71.6 83.5

Example 6: Assessment of Feasibility of High Concentration Formulation

The clinical need for subcutaneous (SC) administration of high doseprotein-based drugs >100 mg/mL oftentimes introduces additionaltechnical development challenges for manufacturing, analytical testing,stability, and delivery. A common attribute of a high concentrationprotein formulation is high viscosity, which results directly from thereversible self-association of proteins. A high viscosity may alsointroduce additional clinical development challenges due to highinjection forces, increase in pain at the injection site, and may alsoalter drug pharmacokinetic profiles. Thus, an important element ofproduct development efforts seek to identify a formulation with a lowviscosity. The effect to viscosity may be mitigated by changes in pH oraddition of excipients.

Due to the expected exponential increase in viscosity as a result ofincrease in mAb concentration up to 200 mg/mL, an initial feasibilitystudy was conducted to investigate the viscosity and injectability of ahigh concentration anti-IL13 solution. A ˜150 mg/mL solution wasconcentrated to ˜210 mg/mL in the previously established acetate basedformulation. Viscosity measurements were taken at the following mAbconcentrations: 50, 150 and 200 mg/mL using a cone and plate rheometer.

FIG. 17 displays the various viscosity levels plotted against theaforementioned concentrations, with an observed exponential increase inviscosity with increasing concentration. The viscosity result for theconcentrated solution at 207.7 mg/mL was 28.6 cP.

The maximum injection force was determined by placing 207.7 mg/mLanti-IL13 in a 1 mL glass syringe fitted with a 27 gauge needle withsyringe speed set at 3 mm/min measured using Instron electro mechanicaltesting system. Table 17 shows the numeric results obtained for bothviscosity and syringeability at the maximum concentration of 207.7mg/mL.

The determination of the viscosity (28.6 centipoise) and maximuminjection force of 30.3 Newtons at the concentration of 207.7 mg/mLprompted that additional formulation development efforts were requiredto enable and achieve the feasibility of manufacturing and dosing a highconcentration anti-IL13 presentation.

TABLE 17 Summary of High Concentration Feasibility DeterminationConcentration 207.7 mg/mL Viscosity 28.6 centipoise Maximum InjectionForce 30.3 Newtons

Example 7: Assessment of Physical Properties Via Shake Studies

Histidine and succinate buffer at pH 6.25 were identified as the optimalbuffer systems, however additional formulation development studies wererequired to identify suitable formulation for high concentration mAbsolutions susceptible to high viscosity dependent on high concentrationand gelation as a function of temperature.

The formulations used in the shake studies stemmed from the HTFscreening study. The HTF study identified two buffer systems (histidineand succinate at pH 6.25) that provided good stability. A third buffersystem (50 mM acetate pH 5.5) was also included as a control. Thehistidine and succinate pH 6.25 buffer system samples were preparedusing small scale buffer exchange and concentration techniques. Thefollowing excipients were then added: 0.02% polysorbate 80 (PS80) toprotect the protein from shear stress, and 150 mM sodium chloride as apotential viscosity lowering agent.

The samples were filled at 1.2 mL volume in 3 mL glass vial at a 1 mLfill volume and were shaken for 72 hours at 250 rpm at 2-8° C. on ahorizontal shaker protected from light. The samples were then tested byvarious analytical techniques. Table 18 below lists the formulationsused for the shear stress/shake study. Lower concentration formulationswere included as no previous shake study with 50 mg/mL had beenattempted. Control formulations with no excipients such as PS80 werealso included. For the acetate samples, NaCl controls were included.

TABLE 18 Shake study design and samples. Formu- mAb Surfac- Viscositylation Sample conc. Buffer tant modifier # type (mg/mL) system pH(0.02%) (150 mM) 1 Low 50 50 mM 5.5 PS80 NaCl mAb Sodium Acetate 2 50 20mM 6.25 PS80 NaCl Sodium Succinate 3 50 20 mM 6.25 PS80 NaCl Histidine 4High 200 50 mM 5.5 PS80 NaCl mAb Sodium Acetate 5 200 20 mM 6.25 PS80NaCl Sodium Succinate 6 200 20 mM 6.25 PS80 NaCl Histidine 7 Buffer 0 50mM 5.5 Controls Sodium Acetate 8 0 20 mM 6.25 Sodium Succinate 9 0 20 mM6.25 Histidine 10 NaCl 200 50 mM 5.5 NaCl Control Acetate

No significant changes were observed in the general appearance, SEC, DLSand MFI in all samples formulated with NaCl and polysorbate 80. Highconcentration protein formulations were not stable in the Acetate(Control) formulation via SEC-HPLC results.

The target concentration of all tested anti-IL13 mAb is 200 mg/mL,however, due to limitations of process yields and inherent variabilityof the viscosity measurements, the nominal concentration formulations inhistidine and succinate at 200 mg/mL is within 10% range. Measurement ofthe viscosity of samples used in the shake study reveal that a 6 foldlower viscosity is achievable in formulations containing 150 mM SodiumChloride at a nominal concentration of 200 mg/mL, regardless of thebuffer system (FIG. 18). Formulations containing the buffers alonedisplayed significantly higher viscosity in Histidine compared to SodiumAcetate and Sodium Succinate. This suggested that the sodium ions of thelatter buffers may be contributing toward the reduction of theviscosity, thus also explaining that the sodium ions of Sodium Chloridemay be responsible for the viscosity lowering effect. However, asHistidine is combined with 150 mM Sodium Chloride, the viscosity islowered to the same degree as in other buffer systems. This may alsosuggest that the there is a synergistic effect between the histidinebuffer and NaCl which is effectively lowering the viscosity.

This study also included formulations 1, 2, and 3 at a lowerconcentration of 50 mg/mL; however, no difference was observed among theformulations, indicating the importance of generated stability data forhigh concentration formulation at the target concentration (data notshown).

The dependence of viscosity on protein concentration and sodium contentof the samples is summarized in FIG. 18. The viscosity readings for 50mg/mL (lower concentration) samples were all less than 2 cps. Theviscosity of acetate sample with a nominal concentration of 200 mg/mLcontaining 150 mM NaCl was about 3 cps lower than the correspondinghistidine and succinate samples, primarily due to the lower than nominalconcentration of 200 mg/mL due to poor process yields. The overallconcentrations were less than 200 mg/mL, due to losses during theconcentrated sample preparation as shown in FIG. 18.

The results of the study clearly indicated that inclusion of 150 mM NaClsubstantially reduced viscosity across the tested buffer systems. Also aunique insight into the synergistic relationship between the histidinebuffer and NaCl was observed.

Note: The target concentration of all tested anti-IL13 mAb is 200 mg/mL,however, due to limitations of process yields and inherent variabilityof the viscosity measurements, the nominal concentration formulations inhistidine and succinate at 200 mg/mL is within ±10% range.

Example 8: Assessment of Physical Properties at Elevated Temperatures

The following study was designed to assess the physical stability ofhigh concentration anti-IL13 at a nominal concentration of 200 mg/mLupon exposure to thermal stress at suitably identified elevatedtemperature.

Samples were incubated at an elevated temperature of 50° C./60% RH for 7or 10 days. Analytical testing included general appearance (GA),viscosity, concentration by A280 nm, SEC-HPLC, MFI and DLS. Table 19summarizes the sample formulations used for the elevated temperaturestudy.

TABLE 19 Formulations used for elevated temperature study Surfac-Viscosity Formula- mAb conc. tant modifier tion# (mg/mL) Buffer systempH (0.02%) (150 mM) 1 200 50 mM Acetate 5.5 PS80 NaCl 2 200 20 mMSuccinate 6.25 PS80 NaCl 3 200 20 mM Histidine 6.25 PS80 NaCl

The general appearance results, as summarized in Table 20 and FIG. 19,revealed gelation of formulation 1 (Acetate) after 7 days. Formulation 2(Succinate) displayed semi-gelation after 10 days storage, whileformulation 3 (Histidine) displayed no gelation of the sample after 10days storage. No particles were observed in any of the samples which didnot gel. The 7 days and 10 days succinate and histidine samples wereclassified as “milky iridescent.” Milky iridescent equates to decreasein transparency which could be indicative of a higher concentration ofsubvisible particles and aggregation. MFI results (not shown) revealedt=0 of formulation 1 (Acetate) had ˜50% more particles than otherformulations.

TABLE 20 GA results for the planned 2 wk elevated temperature study at50° C./60% RH Time Formulation (days) General Appearance (GA)Formulation 1: 0 Opalescent Acetate 7 Gel, milky iridescent 10 Geldarker and more solid and opaque than 7 day gel Formulation 2: 0Opalescent Succinate 7 Milky iridescent (more opalescent than initial)10 Semi-gel (gel could move within vial), milky Formulation 3: 0Opalescent Histidine 7 Milky iridescent 10 Milky iridescent solution

The results of the study clearly indicated the combination of 150 mMNaCl with histidine buffer at pH 6.25 prevented the gelation phenomena.The results also indicated that buffer type had a significant impact onphysical stability (Histidine>Succinate>Acetate). These results maysuggest a synergistic relationship which exists between the histidinebuffer and NaCl to enhance the physical stability of high concentrationmAb formulations.

Example 9: 200 mg/mL Freeze-Thaw Study

The purpose of this study was to evaluate the impact of threefreeze-thaw cycles ranging between 2-8° C. and −70° C. on the physicaland chemical stability profile of anti-IL13 mAb at a nominalconcentration of 200 mg/mL. The freeze-thaw study examined stability in15 mM histidine buffer pH 6.25, containing either 100 or 150 mM NaCl,0.05 mm EDTA, 0.02% polysorbate 80. An acetate based formulation wasincluded as a control at pH 5.5. The samples were filled at 1.2 mL fillvolume into 3 mL glass vials and subjected to three freeze-cyclesbetween 2-8° C. and −70° C., protected from light. Table 21 displays thesamples for this study.

TABLE 21 Sample formulations tested for the freeze-thaw studyFormulation 200 mg/mL antiIL 13 in 15 mM Histidine A Control pH 6.25 +0.05 mM EDTA + 0.02% F/T -A 3 Freeze/ polysorbate 80 + 100 mM NaCl Thawcycles 200 mg/mL anti-IL13 in 15 mM Histidine B Control pH 6.25 + 0.05mM EDTA + 0.02% F/T -B 3 Freeze/ polysorbate 80 + 150 mM NaCl Thawcycles 200 mg/mL anti-IL13 in a acetate based C Control formulation withvarious excipients at F/T -C 3 Freeze/ pH 5.5 Thaw cycles

The samples were analyzed with the following analytical techniques: GA,pH, viscosity, concentration by A280 nm, potency by SPR, SDS-PAGE,SEC-HPLC, cIEF and MFI.

Freeze Thaw (F/T). There were no significant differences between resultsfor F/T and control samples for any formulation by general appearance,pH, potency by SPR, concentration, SEC, SDS-PAGE or viscosity. There wasno decrease in cIEF % Main from initial for the histidine formulations.Table 22 lists all the general appearance, pH, potency, concentrationand viscosity data. Table 23 lists all the SDS-PAGE data. Table 24 listsall SEC and cIEF data.

The overall results of the study clearly indicated the combination of150 mM NaCl with histidine buffer at pH 6.25 was the best formulationand stable after being subjected to the freeze-thaw stress.

TABLE 22 Analytical testing results for freeze-thaw by GA, pH, potency,concentration and viscosity Potency Concen- Sample General (Biacore)tration Viscosity Description Appearance pH (mg/mL) (mg/mL) (cps) AOpalescent, 6.16 191.4 197.5 17.9 BY4, practically free from visibleparticles F/T -A Opalescent 6.22 197.8 195.1 17.7 BY4, practically freefrom visible particles B Opalescent 6.24 190.0 196.5 16.6 BY4,practically free from visible particles F/T -B Opalescent, 6.24 197.3201.0 16.6 BY4, practically free from visible particles C Opalescent,5.57 166.8 186.5 19.5 BY5, practically free from visible particles F/T-C Opalescent, 5.57 171.3 199.1 18.9 BY5, practically free from visibleparticles

TABLE 23 Freeze-Thaw Study Results (SDS-PAGE) Sample Non reduced ReducedDescrip- % % % % % % tion Main Agg Frag Heavy Light H + L A 82.8 0.810.7 64.8 32.3 97.1 F/T -A 82.4 0.6 11.1 65.6 31.2 96.8 B 83.6 0.7 10.365.0 31.5 96.6 F/T -B 83.6 0.6 10.4 65.8 31.0 96.8 C 80.1 1.1 12 63.332.0 95.2 F/T -C 78.8 1.2 12.9 63.9 31.5 95.4

TABLE 24 Freeze-Thaw Study Results (SEC, cIEF) SEC Sample % % cIEFDescrip- Total Mono- % # pI % % % tion Agg mer Frag bands main MainAcidic Basic A 2.5 97.5 0.1 7 7.68 70.7 9.5 19.9 F/T -A 2.5 97.4 0.0 77.68 72.8 9.2 18.1 B 2.4 97.5 0.1 7 7.68 71.2 8.9 20.1 F/T -B 2.4 97.50.1 7 7.68 73.0 7.7 19.3 C 2.1 97.9 0.1 7 7.68 65.9 12.1 22.1 F/T -C 2.197.8 0.1 7 7.68 65.6 12.0 22.5

Example 10: Short Term Chemical Stability Study and BiophysicalCharacterization of a mAb Based Drug Product at a Nominal Concentrationof 200 mg/mL

A development stability study was performed to assess the short-termchemical stability of a 200 mg/mL mAb formulation containing NaCl, PS80and EDTA. The short term chemical stability study examined stability in15 mM histidine buffer pH 6.25, containing either 100 or 150 mM NaCl,0.05 mm EDTA, 0.02% polysorbate 80. An acetate based formulation wasincluded as a control. The samples were filled at 1.2 mL fill volumeinto 3 mL glass vials and placed at the following storage conditions 5°C., 25° C. and 40° C. Table 25 below displays the samples for thisstudy.

TABLE 25 Short Term Chemical Stability Samples and initial osmolalityresults Formula Osmolality Formulations code (mOsm) 200 mg/mL anti-IL13in 15 mM Histidine pH A 216 6.25 + 0.05 mM EDTA + 0.02% Polysorbate80 +100 mM NaCl 200 mg/mL anti-IL13 in 15 mM Histidine pH B 333 6.25 + 0.05mM EDTA + 0.02% Polysorbate80 + 150 mM NaCl 200 mg/mL anti-IL13 in 50 mMacetate pH 5.5 + C 293 51 mM NaCl + 0.05 mM EDTA + 1% arginine + 0.02%Polysorbate80

The samples were analyzed with the following analytical techniques: GA,pH viscosity, concentration by A280 nm, potency by SPR, SDS-PAGE,SEC-HPLC and cIEF. The study was for a total duration of 3 months andvarious biophysical characterization testing was also performed atinitial and final time-point with DSC, DLS, CD and MFI.

Results from analytical (concentration, potency, SEC, SDS-PAGE, cIEF)and biophysical characterization (DSC, fluorescence, CD) testingindicate that formulation B (200 mg/mL anti-IL13 in 15 mM histidine, pH6.25, 150 mM NaCl, 0.05 mM EDTA, 0.02% polysorbate 80) was the best ofthe three formulations for short term chemical stability.

Analytical Testing Results Summary and Discussion:

Table 26 summarizes 5° C. data up to 3 months by all the analyticaltechniques and Table 27 summarizes the stressed and accelerated data at25° C. and 40° C. up to 3 months.

Samples in histidine formulations (A and B) exhibited higher % mainafter 3 months at 5° C. and 25° C. than the acetate control samples, asdetermined by cIEF. Purity determined by SDS-PAGE revealed higher %Heavy and Light chain content for samples in both histidine formulations(A and B) after 3 months at 25° C., than acetate control samples. Atstorage condition 40° C./75% RH, all three formulations after 1 month,exhibited a lower % H+L content compared to the initials, howeverformulations A and B surpassed formulation C (90.6 and 91.8 versus88.2%). There were significant decreases in % Main for all 1 month 40°C. samples, but the decreases were smaller for the histidineformulations (8.8 or 7.5%) versus the change for acetate formulation(9.9%), with formulation B showing the lowest % decrease. Highestpotency results were observed in histidine based formulation Bcontaining 150 mM NaCl at the 3 months time-point at 25° C./60% RH and 1month time point at 40° C./75% RH. Lowest potency was observed in thecontrol and former acetate based formulation C which is not providingsuitable stability for the high concentration mAb. Viscosity results forformulations A and B initial testing (18.0 and 16.3 cps) were lower thanfor formulation C (18.7 cps), as expected due to the higher NaClconcentration in formulations A and B (100 and 150 mM NaCl,respectively). Formulation B viscosity results were the lowest at allconditions and time points.

TABLE 26 5° C. Data for Short Term Chemical Stability Formulation AssayCode Initial 1 m 5° C. 3 m 5° C. 16 m 5° C. General A Opalescent,Opalescent, Opalescent, Opalescent Appearance between B5 and B4, BY4,practically BY4, practically BY4, essentially free from visible freefrom visible free from visible free from visible particles particlesparticles particles B Opalescent, Opalescent, Opalescent, Opalescent.between B5 and B4, BY4, practically BY4, practically BY4, essentiallyfree from visible free from visible free from visible free from visibleparticles particles particles particles C Opalescent, Opalescent,Opalescent, Opalescent, between B5 and B4, BY5, practically BY5,practically BY5, essentially free from visible free from visible freefrom visible free from visible particles particles particles particlespH A 6.24 6.23 6.21 6.20 B 6.26 6.24 6.24 6.19 C 5.55 5.58 5.64 5.60Potency A 197.6 197.5 190.0 NT B 194.2 194.2 185.0 NT C 173.9 171.8170.0 NT Concentration A 202.6 196.12 195.0 196.2 (mg/mL) B 202.6 196.94198.9 198.1 C 208.2 190.46 185.7 199.4 SDS-PAGE A NT 97.5 97.0 97.9 (%H + L, reduced) B NT 97.6 97.0 97.7 C NT 96.2 96.0 96.0 SEC % TotalAgg/A 2.4 97.6 0.0 3.0 96.9 0.1 2.6 97.3 0.1 3.0 96.9 0.1 % Monomer/ B 2.397.7 0.0 3.0 96.9 0.1 2.5 97.4 0.1 3.0 96.9 0.1 % Fragment C 2.0 98.00.0 2.1 97.8 0.1 2.0 97.9 0.1 2.1 97.8 0.1 cIEF A 7.67 70.9 7.68 71.47.68 73.4 7.68 73.6 pI/% Main B 7.67 70.6 7.68 71.8 7.68 73.9 7.68 73.6C 7.68 63.7 7.68 65.0 7.68 66.5 7.68 67.4 Viscosity A 18.0 17.6 18.415.87 (cps) B 16.3 16.3 17.0 14.85 C 18.7 18.5 18.8 16.41

TABLE 27 Stressed and Accelerated Data for Short Term Chemical StabilityFormulation Assay Code Initial 1 m 40° C. 3 m 25° C. General AOpalescent, Opalescent, Opalescent, Appearance between B5 and 84, BY4,practically BY4, practically free from visible free from visible freefrom visible particles particles particles B Opalescent, Opalescent,Opalescent, between B5 and B4, BY4, practically BY4, practically freefrom visible free from visible free from visible particles particlesparticles C Opalescent, Opalescent Opalescent, between B5 and B4, BY5,practically BY5, practically free from visible free from visible freefrom visible particles particles particles pH A 6.24 6.18 6.20 B 6.266.22 6.22 C 5.55 5.51 5.66 Potency A 197.6 138.1 167.0 B 194.2 149.0176.0 C 173.9 124.8 146.0 Concentration A 202.6 195.12 185.1 (mg/mL) B202.6 199.04 195.1 C 208.2 187.39 200.3 SDS-PAGE A NT 90.6 95.7 (% H +L, reduced) B NT 91.8 95.3 C NT 88.2 93.1 SEC % TotalAgg/ A 2.4 97.6 0.05.3 92.9 1.8 3.6 94.7 1.7 % Monomer/ B 2.3 97.7 0.0 5.2 93.1 1.7 3.694.9 1.5 % Fragment C 2.0 98.0 0.0 4.8 93.5 1.8 2.6 95.6 1.9 cIEF A 7.6770.9 7.68 62.1 7.68 70.9 pI/% Main B 7.67 70.6 7.68 63.1 7.68 69.8 C7.67 63.7 7.68 53.8 7.68 60.6 Viscosity A 18.0 18.2 19.1 (cps) B 16.317.2 16.5 C 18.7 20.0 19.3

Biophysical Characterization Results Summary and Discussion:

Table 28 shows DLS data for initial and Fluorescence data for initialand 3 months.

MFI results displayed no visible particles were observed in any sampleover 3 months. No significant differences were observed among the threeformulations by DLS, which measure the hydrodynamic radii of the mAb.Fluorescence and circular dichroism data did not indicate perturbationsto the secondary and tertiary structure in histidine formulations after3 months storage. FIG. 20 displays the CD comparison plot of thespectra. The lower signal for formulation C samples could indicate achange in the tertiary structure. The differences seen in % quench forformulation C also indicate a change in tertiary structure for thisformulation. The DSC data for initial and 3 month samples are displayedin Table 29. The trend of higher Tm and total kcal/mole results for bothhistidine/NaCl formulations suggests that the additional salt allows forgreater thermodynamic stability than the acetate formulation.

TABLE 28 DLS and Fluorescence Data for Short Term Chemical Stability DLSFluorescence Rh max F.I. at max Quenching Sample Condition (nm) (nm)(absorbance) (%) A Initial 5.5 340 402 n/a B Initial 5.5 342 451 n/a CInitial 5.7 341 365 n/a A 3 m 5° C. NT 341 418 4.0 B 3 m 5° C. NT 341454 0.7 C 3 m 5° C. NT 342 466 27.7 A 3 m 25° C. NT 341 352 −12.4 B 3 m25° C. NT 341 407 −9.8 C 3 m 25° C. NT 342 452 23.8 A 16 m 5° C. NT 340355 −11.7 B 16 m 5° C. NT 341 402 −10.9 C 16 m 5° C. NT 342 436 19.5

TABLE 29 DSC Data for Short Term Chemical Stability Tm1 Tm2 Tm3 Totalarea Formula Code (° C.) (° C.) (° C.) kcal/mole A Initial 68.7 74.082.8 960 A 3 m 5° C. 68.6 74.0 82.8 988 A 3 m 25° C. 68.5 74.5 82.9 1122A 16 m 5° C. 68.8 74.3 82.8 902 B Initial 68.4 73.4 82.7 1036 B 3 m 5°C. 68.4 73.4 82.6 965 B 3 m 25° C. 68.3 73.8 82.7 1055 B 16 m 5° C. 68.473.5 82.6 1081 C Initial 67.9 73.1 82.9 884 C 3 m 5° C. 67.7 73.1 82.9891 C 3 m 25° C. 67.7 73.6 83.0 947 C 16 m 5° C. 67.7 73.2 82.8 928

Example 11: Long Term Chemical Stability Study and BiophysicalCharacterization of a mAb Based Drug Product at a Nominal Concentrationof 200 mg/mL

A 16-month time-point was tested for all samples on stability as listedin Table 26. The results of this time point was used to assess thelong-term chemical stability of the mAb drug product at a nominalconcentration of 200 mg/mL formulated in His, NaCl, PS80 and EDTA.Limited testing was performed to confirm the long-term stability.

The samples were analyzed with the following analytical techniques: GA,pH, viscosity, concentration by A280 nm, SDS-PAGE, SEC-HPLC and cIEF.Biophysical characterization testing was also performed which includedfluorescence, DSC and CD.

Table 26 includes 5° C. data at the 16 months time-point by all theanalytical techniques.

Analytical testing results summary and discussion: Samples in histidineformulations (A and B) exhibited higher % Monomer content after 16months at 5° C. Formulation C was 0.9% higher than either of thehistidine formulations.

Biophysical characterization results summary and discussion: Overall,biophysical characterization results were comparable between the 3 monthand the 16 months time-point. Fluorescence and circular dichroism datadid not indicate perturbations to the secondary and tertiary structurein histidine formulations after 16 months storage. The results for 16month formulation A and B samples at 5° C. show no significant changes.There were significant intensity increases for both formulation Csamples, which indicates that the molecule is unfolding and exposingmore fluorescent groups. There was no increase in visible particles at16 month time point based on analysis by MFI results for formulations Aand B. The DSC data for samples at 16 months time point. The trend ofhigher Tm and total kcal/mole results for both histidine/NaClformulations suggests that the additional salt allows for greaterthermodynamic stability than the acetate formulation.

Table 28 shows Fluorescence data for 16 month time-point. Table 29 showsthe DSC results for the 16 month time-point.

Results from analytical (concentration, potency, SEC, SDS-PAGE, cIEF)and biophysical characterization (DSC, fluorescence, CD) testingindicate that Formulation A (200 mg/mL anti-IL13 in 15 mM histidine, pH6.25, 150 mM NaCl, 0.05 mM EDTA, 0.02% polysorbate 80) was the best ofthe three formulations for long term chemical stability.

Sequence Listing BLyS SEQ ID NO: 1MDDSTEREQS RLTSCLKKRE EMKLKECVSI LPRKESPSVRSSKDGKLLAA TLLLALLSCC LTVVSFYQVA ALQGDLASLRAELQGHHAEK LPAGAGAPKA GLEEAPAVTA GLKIFEPPAPGEGNSSQNSR NKRAVOGPEE TVTQDCLQLI ADSETPTIQKGSYTFVPWLL SFKRGSALEE YENKTLVYET GYFFIYGQVLYTDKTYAMGH LIQRKEVBVF GDELSLVTLF RCIQNMPETLPNNSCYSAGI AKLEEGDELQ LAIPRENAQI SLDGDVTFFG ALKLL Belimumab VHSEQ ID NO: 2 QVQLQQSGAE VKKPGSSVRV SCKASGGTFN NNAINWVRQAPGQGLEWMGG IIPMFGTAKY SQNFQGRVAI TADESTGTASMELSSLRSED TAVYYCARSR DLLLFPHHAL SPWGRGTMVT VSS Belimumab VLSEQ ID NO: 3 SSELTODPAV SVALGQTVRV TCQGDSLRSY YASWYQQKPGQAPVLVIYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAEDEADYYCSSR DSSGNHWVEG GGTELTVLG Tabalumab VH SEQ ID NO: 4MKHLWFFLLL VAAPRWVLSQ VQLQQWGAGL LKPSETLSLTCAVYGGSFSG YYWSWIRQPP GKGLEWIGEI NHSGSTNYNPSLKSRVTISV DTSKNQFSLK LSSVTAADTA VYYCARGYYD ILTGYYYYFD YWGQGTLVTV SSTabalumab VL SEQ ID NO: 5 EIVLTQSPAT LSLSPGERAT LSCRASQSVS RYLAWYQQKPGQAPRLLIYD ASNRATGTPA RFSGSGSGTD STLTISSLEPEDFAVYYCQQ RSNWPRTFGQ GTKVEIKRT Belimumab heavy chain SEQ ID NO: 6QVQLOQSGAE VKKPGSSVRV SCKASGGTFN NNAINWVRQAPGQGLEWMGG IIPMFGTAKY SQNFQGRVAI TADESTGTASMELSSLRSED TAVYYCARSR DLLLFPHHAL SPWGRGTMVTVSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPVTVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLGTQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPELLGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMBEALHN HYTQKSLSLS PGSBelimumab light chain SEQ ID NO: 7SSELTQDPAV SVALGQTVRV TCQGDSLRSY YASWYQQKPGQAPVLVTYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAEDEADYYCSSR DSSGNHWVFG GGTELTVLGQ PKAAPSVTLFPPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAGVETTTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECSTabalumab heavy chain SEQ ID NO: 8QVQLQQWGAG LLYPSETLSL TCAVYGGSFS GYYWGWIRQPPGKGLEWIGE INHSGSTNYN PSLKSRVTIS VDTSKNQFSLKLSSVTAADT AVYYCARGYY DILTGYYYYF DYWGQGTLVTVSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPVTVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLGTKTYTCNVDH KPSOTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNWYVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSISISLGKTabalumab light chain SEQ ID NO: 9EIVLTQSPAT LSLSPGERAT LSCRASQSVS RYLAWYQQKPGQAPRLLIYD ASNRATGIPA RFSGSGSGTD STLTISSLEPEDFAVYYCQQ RSNWPRTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSNTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGESSoluble Form of BLyS SEQ ID NO: 10AVQGPEETVT QDCLQLIADS ETPTIQKGSY TFVPWILSFKRGSALEEKEN KILVKETGYF FIYGQVLYTD KTYAMGHLIQREKVHVEGDE LSLVTLFRCI QNMPETLPNN SCYSAGIAKLEEGDELQLAI PRENAQISLD GDVTFFGALK LL Belimumab CDRH1 SEQ ID NO: 11GGTFNNNAIN Belimumab CDRH2 SEQ ID NO: 12 GIIPMFGTAK YSQNFQGBelimumab CDRH3 SEQ ID NO: 13 SRDLLLFPHH ALSP Belimumab CDRL1SEQ ID NO: 14 QGDSLRSYYA S Belimumab CDRL2 SEQ ID NO: 15 GKNNRPSBelimumab CDRL3 SEQ ID NO: 16 SSRDSSGNHW V

1. A pharmaceutical formulation for an antigen binding proteincomprising: a. about 150 to 250 mg/mL antigen binding protein; b. about1 to 100 mM of a buffering agent providing a pH of 6.0±0.5; c. about 90to 150 mM of a tonicity agent; d. about 1 to 100 mM of a stabilizer; ande. about 0.005 to 0.015% (w/v) of a nonionic surfactant: wherein theantigen binding protein comprises amino acid sequences CDRH1 of SEQ IDNO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ IDNO: 14, CDRL2 of SEQ ID NO:15, and CDRL3 of SEQ ID NO:16. 2-3.(canceled)
 4. The pharmaceutical formulation according to claim 1,wherein said antigen binding protein is a monoclonal antibody orfragment thereof.
 5. The pharmaceutical formulation according to claim4, wherein said monoclonal antibody or fragment thereof binds to BLyS(SEQ ID NO: 1) or a hetero- or homo-trimeric form of BLyS.
 6. Thepharmaceutical formulation according to claim 4, wherein said monoclonalantibody comprises heavy and light chain variable regions comprisingamino acid sequences that are 90% identical to SEQ ID NOs: 2 and 3,respectively.
 7. The pharmaceutical formulation according to claim 4,wherein said monoclonal antibody comprises heavy and light chainscomprising amino acid sequences that are 90% identical to SEQ ID NOs: 6and 7, respectively.
 8. The pharmaceutical formulation according toclaim 4, wherein the monoclonal antibody concentration is 200±20 mg/mL.9. The pharmaceutical formulation according to claim 4, wherein thetonicity agent is sodium chloride.
 10. The pharmaceutical formulationaccording to claim 9, wherein the sodium chloride concentration is115±10 mM.
 11. The pharmaceutical formulation according to claim 4,wherein the buffering agent is histidine.
 12. The pharmaceuticalformulation according to claim 11, wherein the histidine concentrationis about 5 to 15 mM.
 13. The pharmaceutical formulation according toclaim 12, wherein the histidine concentration is 10±2 mM. 14-15.(canceled)
 16. The pharmaceutical formulation according to claim 4wherein the stabilizer is arginine.
 17. The pharmaceutical formulationaccording to claim 16, wherein the arginine concentration is about 20 to30 mM.
 18. The pharmaceutical formulation according to claim 17, whereinthe arginine concentration is 25±2 mM.
 19. (canceled)
 20. Thepharmaceutical formulation according to claim 4, wherein the nonionicsurfactant is polysorbate
 80. 21. The pharmaceutical formulationaccording to claim 20, wherein the polysorbate 80 concentration is about0.01% (w/v).
 22. The pharmaceutical formulation according to claim 4which is stable upon freezing and thawing.
 23. (canceled)
 24. Thepharmaceutical formulation according to claim 4 for subcutaneous orintramuscular administration.
 25. The pharmaceutical formulationaccording to claim 8 wherein: the tonicity agent is sodium chloride at aconcentration of 115±10 mM; the buffering agent is histidine at aconcentration of 5 to 15 mM; the stabilizer is arginine at aconcentration of 20 to 30 mM; and the nonionic surfactant is polysorbate80. 26-30. (canceled)
 31. A method of treating a disease or conditionwhich is amenable to treatment with an anti-BLyS antibody in a humancomprising administering to said human a formulation according to claim4, wherein said disease or condition is selected from the groupconsisting of systemic lupus erythematosus, anti-neutrophil cytoplasmicantibody vasculitis, lupus nephritis, primary Sjögren's syndrome,chronic immune thrombocytopenia, myasthenia gravis, symptomaticWaldenström's macroglobulinaemia, immune desensitizing of patientsawaiting kidney transplant, membranous nephropathy, systemic sclerosis,rheumatoid arthritis, multiple myeloma, multiple sclerosis, and kidneyfailure. 32-34. (canceled)